Mucin 5b (muc5b) irna compositions and methods of use thereof

ABSTRACT

The present invention relates to RNAi agents, e.g., dsRNA agents, targeting the mucin 5B (MUC5B) gene. The invention also relates to methods of using such RNAi agents to inhibit expression of a MUC5B gene and to methods of treating or preventing a MUC5B-associated disease, such as a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF), in a subject.

RELATED APPLICATIONS

This application is a 35 § U.S.C. 111(a) continuation application which claims the benefit of priority to PCT/US2021/056131, filed on Oct. 22, 2021, which, in turn, claims the benefit of priority to U.S. Provisional Application No. 63/104,557, filed on Oct. 23, 2020. The entire contents of each of the foregoing applications are incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML copy, created on Sep. 8, 2023, is named 121301_13102_SL.xml and is 25,830,600 bytes bytes in size.

BACKGROUND OF THE INVENTION

Mucin 5B (MUC5B) is a member of the mucin family of proteins, which are highly glycosylated macromolecular components of mucus secretions. This family member is expressed in club cells in normal lung epithelium. The MUC5B protein is the major gel-forming mucin in mucus which is secreted by submucosal glands, salivary glands, nasal mucosa, gallbladder, submucosal glands in trachea and esophagus. It is a major contributor to the lubricating and viscoelastic properties of whole saliva, normal lung mucus and cervical mucus and, in the lung, the MUC5B protein plays a key role in mucociliary transport and mucociliary clearance (MCC) and host defense responsible for trapping and clearing inhaled particles.

Increased expression of MUC5B is associated with the development of lung diseases, e.g., pulmonary fibrosis, cystic fibrosis, and/or chronic obstructive pulmonary disease (COPD). For example, mice overexpressing Muc5b exhibit increased mucosal depth, reduced ciliary beat frequency, and reduced mucociliary transport rate and bleomycin-treated mice overexpressing Muc5b in distal airways and alveoli have decreased survival and increased lung fibrosis. Furthermore, increased MUC5B expression has been onserved in the distal airways of subjects having idiopathic pulmonary fibrosis (IPF).

IPF is the result of repeated injury to the alveolar epithelium in the lung resulting in a pro-inflammatory and fibroproliferative response. IPF, affecting 5 million subjects worldwide, is a progressive lung disease characterized by a pattern of heterogeneous, subpleural patches of fibrotic, remodeled lung and honeycomb cysts. Once diagnosed with IPF, a subject has a median survival of 3-5 years. Excess production of MUC5B disrupts the normal reparative mechanisms and decreases mucociliary clearance which leads to retention of damaging particles and, thus, repeated injury, pro-inflammatory and fibroproliferative responses, and ultimately to IPF.

IPF is a complex disease caused by both genetic and environmental factors. Risk factors include male gender, age, smoking status, and certain occupational exposures (e g, mining, farming, construction). However, the strongest risk factor for IPF, genetic or otherwise, is a pathogenic gain-of-function variant (rs35705950) in the promoter of the MUC5B gene, resulting in increased expression of MUC5B, and accounting for ˜30% of total risk. Heterozygosity for rs35705950 is associated with a 6-fold increase in IPF and homozygosity is associated with a 20-fold increase in IPF. Importantly, increased MUC5B expression in distal airways and in type 2 alveolar epithelial cells and columnar epithelial cells lining honeycomb cysts has been observed in IPF subjects that do not carry the rs35705950 variant.

Current therapies (e.g., Pirfenidone, Nintedanib) are mainly supportive and may slow the progression of disease but have little impact on overall survival.

Accordingly, there exists an unmet need for effective treatments for lung diseases, e.g., pulmonary fibrosis, e.g., IPF; cystic fibrosis, and/or chronic obstructive pulmonary disease (COPD), such as an agent that can selectively and efficiently silence the MUC5B gene using the cell's own RNAi machinery that has both high biological activity and in vivo stability, and that can effectively inhibit expression of a target MUC5B gene.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides RNAi agent compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcipts of a gene encoding Mucin 5B (MUC5B). The MUC5B gene may be within a cell, e.g., a cell within a subject, such as a human. The present disclosure also provides methods of using the RNAi agent compositions of the disclosure for inhibiting the expression of a MUC5B gene or for treating a subject who would benefit from inhibiting or reducing the expression of a MUC5B gene, e.g., a subject having a MUC5B-associated disorder, e.g., a subject having a lung disease, e.g., pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF); cystic fibrosis, and/or chronic obstructive pulmonary disease (COPD), or a subject at risk of developing a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis, e.g., a subject carrying the rs35705950 variant (GT or TT).

Accordingly, in one aspect, the instant disclosure provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a Mucin 5B (MUC5B) gene, wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:6; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In one aspect, the present invention provides a double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of a MUC5B gene in a cell, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises a region complementary to part of an mRNA encoding a MUC5B gene (SEQ ID NO:1), wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In yet another aspect, the present invention provides a double stranded RNAi agent for inhibiting expression of a a MUC5B gene in a cell, comprising a sense strand and an antisense strand forming a double stranded region, wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-7, 9, and 10, wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.

In one embodiment, the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any of the sense strands and antisense strands in any one of Tables 2-7, 9, and 10.

In one embodiment, the antisense strand and/or the sense strand comprises at least 15 contiguous nucleotides differing by no more than three nucleotides from the antisense strand and/or the sense strand nucleotide sequences of a duplex selected from the group consisting of AD-1318337, AD-1318338, AD-1314054, AD-1317692, and AD-1318239.

In one embodiment, both the sense strand and the antisense strand is conjugated to one or more lipophilic moieties.

In one embodiment, lipophilicity of the lipophilic moiety, measured by log Kow, exceeds 0.

In one embodiment, the hydrophobicity of the double-stranded RNAi agent, measured by the unbound fraction in a plasma protein binding assay of the double-stranded RNAi agent, exceeds 0.2.

In one embodiment, the plasma protein binding assay is an electrophoretic mobility shift assay using human serum albumin protein.

In one embodiment, the dsRNA agent comprises at least one modified nucleotide.

In some embodiments, substantially all of the nucleotides of the antisense strand are modified nucleotides.

In another embodiment, all of the nucleotides of the sense strand and all of the nucleotides of the antisense strand comprise a modification.

In one embodiment, at least one of the modified nucleotides is selected from the group a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-hydroxyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA)S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, a 2′-O hexadecyl nucleotide, a nucleotide comprising a 2′-phosphate, a cytidine-2′-phosphate nucleotide, a guanosine-2′-phosphate nucleotide, a 2′-O-hexadecyl-cytidine-3′-phosphate nucleotide, a 2′-O-hexadecyl-adenosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-guanosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-uridine-3′-phosphate nucleotide, a a 5′-vinyl phosphonate (VP), a 2′-deoxyadenosine-3′-phosphate nucleotide, a 2′-deoxycytidine-3′-phosphate nucleotide, a 2′-deoxyguanosine-3′-phosphate nucleotide, a 2′-deoxythymidine-3′-phosphate nucleotide, a 2′-deoxyuridine nucleotide, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof.

In another embodiment, modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, 3′-terminal deoxy-thymine nucleotides (dT), a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a 2′-O-methyl modified nucleotide, a nucleotide comprising glycol nucleic acid (GNA), a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

In another embodiment, the modified nucleotide comprises a short sequence of 3′-terminal deoxy-thymine nucleotides (dT).

In yet another embodiment, the modifications on the nucleotides are 2′-O-methyl modifications, 2′-deoxy-modifications, 2′fluoro modifications, 5′-vinyl phosphonate (VP) modification, and 2′-O hexadecyl nucleotide modifications.

In certain embodiments, the double stranded RNAi agent does not include an inverted abasic nucleotide.

In one embodiment, the dsRNA agent further comprises at least one phosphorothioate internucleotide linkage.

In one embodiment, the dsRNA agent comprises 6-8 phosphorothioate internucleotide linkages.

In one embodiment, each strand is no more than 30 nucleotides in length.

In one embodiment, at least one strand comprises a 3′ overhang of at least 1 nucleotide.

In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides.

The double stranded region may be 15-30 nucleotide pairs in length; 17-23 nucleotide pairs in length; 17-25 nucleotide pairs in length; 23-27 nucleotide pairs in length; 19-21 nucleotide pairs in length; or 21-23 nucleotide pairs in length.

Each strand of the dsRNA agent may be 15-30, 17-20, 19-30 nucleotides in length; 19-23 nucleotides in length; or 21-23 nucleotides in length, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In certain embodiments, the double stranded RNAi agent further includes a lipophilic ligand, e.g., a C16 ligand, conjugated to the 3′ end of the sense strand through a monovalent or branched bivalent or trivalent linker.

In one embodiment, the ligand is

-   -   where B is a nucleotide base or a nucleotide base analog,         optionally where B is adenine, guanine, cytosine, thymine or         uracil.

In other embodiments, the agent further comprises a targeting ligand that targets a liver tissue, e.g., one or more GalNAc derivatives, conjugated to the double stranded RNAi agent via a linker or carrier.

In yet other embodiments, the agents further comprise a lipophilic ligand, e.g., a C16 ligand, conjugated to the 3′ end of the sense strand through a monovalent or branched bivalent or trivalent linker and a targeting ligand that targets a liver tissue, e.g., one or more GalNAc derivatives conjugated to the 3′ end of the sense strand through a monovalent or branched bivalent or trivalent linker.

In one embodiment, one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand via a linker or carrier.

In certain embodiments, the lipophilic moiety is not a cholesterol moiety.

In certain embodiments, the agent further comprises a targeting ligand that targets a liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double stranded RNAi agent via a linker or carrier.

In yet other embodiments, the agents further comprise one or more lipophilic moieties conjugated to one or more internal nucleotide positions, optionally via a linker or carrier and a targeting ligand that targets a liver tissue, e.g., one or more GalNAc derivatives, optionally conjugated to the double stranded RNAi agent via a linker or carrier.

In one embodiment, the internal positions include all positions except the terminal two positions from each end of the at least one strand.

In another embodiment, the internal positions include all positions except the terminal three positions from each end of the at least one strand.

In another embodiment, the internal positions exclude a cleavage site region of the sense strand.

In yet another embodiment, the internal positions include all positions except positions 9-12, counting from the 5′-end of the sense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

In one embodiment, the internal positions include all positions except positions 11-13, counting from the 3′-end of the sense strand. Optionally, the internal positions exclude the cleavage site region of the antisense strand. In certain embodiments, the sense strand is 21 nucleotides in length.

In one embodiment, the internal positions exclude a cleavage site region of the antisense strand.

In one embodiment, the internal positions include all positions except positions 12-14, counting from the 5′-end of the antisense strand. In certain embodiments, the antisense strand is 23 nucleotides in length.

In one embodiment, the internal positions include all positions except positions 11-13 on the sense strand, counting from the 3′-end, and positions 12-14 on the antisense strand, counting from the 5′-end. In certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on the antisense strand, counting from the 5′ end of each strand.

In one embodiment, the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 5, 6, 7, 15, and 17 on the sense strand, and positions 15 and 17 on the antisense strand, counting from the 5′-end of each strand. In certain embodiments, the sense strand is 21 nucleotides in length and the antisense strand is 23 nucleotides in length.

In one embodiment, the positions in the double stranded region exclude a cleavage site region of the sense strand.

In one embodiment, the sense strand is 21 nucleotides in length, the antisense strand is 23 nucleotides in length, and the lipophilic moiety is conjugated to position 21, position 20, position 15, position 1, position 7, position 6, or position 2 of the sense strand or position 16 of the antisense strand.

In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, position position 1, or position 7 of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to position 21, position 20, or position of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to position 20 or position 15 of the sense strand.

In one embodiment, the lipophilic moiety is conjugated to position 16 of the antisense strand.

In one embodiment, the lipophilic moiety is an aliphatic, alicyclic, or polyalicyclic compound.

In one embodiment, the lipophilic moiety is selected from the group consisting of lipid, cholesterol, retinoic acid, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In certain embodiments, the lipophilic moiety is not cholesterol.

In one embodiment, the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne.

In one embodiment, the lipophilic moiety contains a saturated or unsaturated C6-C18 hydrocarbon chain.

In one embodiment, the lipophilic moiety contains a saturated or unsaturated C16 hydrocarbon chain.

In one embodiment, the saturated or unsaturated C16 hydrocarbon chain is conjugated to position 6, counting from the 5′-end of the strand.

In one embodiment, the lipophilic moiety is conjugated via a carrier that replaces one or more nucleotide(s) in the internal position(s) or the double stranded region.

In one embodiment, the carrier is a cyclic group selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl; or is an acyclic moiety based on a serinol backbone or a diethanolamine backbone.

In one embodiment, the lipophilic moiety is conjugated to the double-stranded iRNA agent via a linker containing an ether, thioether, urea, carbonate, amine, amide, maleimide-thioether, disulfide, phosphodiester, sulfonamide linkage, a product of a click reaction, or carbamate.

In one embodiment, the lipophilic moiety is conjugated to a nucleobase, sugar moiety, or internucleosidic linkage.

In one embodiment, the double-stranded RNAi agent further includes a phosphate or phosphate mimic at the 5′-end of the antisense strand. Optionally, the phosphate mimic is a 5′-vinyl phosphonate (VP). When the phosphate mimic is a 5′-vinyl phosphonate (VP), the 5′-terminal nucleotide may have the following structure,

-   -   wherein X is O or S;     -   R is hydrogen, hydroxy, fluoro, or C₁₋₂₀ alkoxy (e.g., methoxy         or n-hexadecyloxy);     -   R^(5′) is ═C(H)—P(O)(OH)₂ and the double bond between the C5′         carbon and R5′ is in the E or Z orientation (e.g., E         orientation); and     -   B is a nucleobase or a modified nucleobase, optionally where B         is adenine, guanine, cytosine, thymine, or uracil.

In certain embodiments, the RNAi agent does not include an inverted abasic nucleotide.

In certain embodiments, the double-stranded RNAi agent does not include a targeting ligand.

In certain embodiments, the double-stranded RNAi agent further includes a targeting ligand that targets a receptor which mediates delivery to a respiratory system tissue, e.g., a lipophilic ligand. In certain embodiments, the targeting ligand is a C16 ligand. In certain embodiments, the lipophilic ligand is not a cholesterol moiety.

In certain embodiments, the RNAi agent is taken up in one or more tissues or cell types including, but not limited to, bronchus, bronchiole, alveoli, epithelium including nasal and respiratory epithelium, ciliated epitheilium, and goblet cells; pneumocytes, both type I and type II, macrophages, peritubular interstitium, mediastinal adipose tissue, pulmonary neuronal plexus, In certain embodiments, cell types include club cells, clara cells, and neutrophils and macrophages, both resident and transient.

In one embodiment, the lipophilic moiety or a targeting ligand is conjugated via a bio-clevable linker selected from the group consisting of DNA, RNA, disulfide, amide, funtionalized monosaccharides or oligosaccharides of galactosamine, glucosamine, glucose, galactose, mannose, and combinations thereof.

In one embodiment, the 3′ end of the sense strand is protected via an end cap which is a cyclic group having an amine, said cyclic group being selected from the group consisting of pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, and decalinyl.

In one embodiment, the dsRNA agent further comprises a targeting ligand that targets a liver tissue.

In one embodiment, the targeting ligand is a GalNAc conjugate.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first internucleotide linkage at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp configuration or Sp configuration.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, second and third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the third internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In one embodiment, the dsRNA agent further comprises a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 3′ end of the antisense strand, having the linkage phosphorus atom in Sp configuration, a terminal, chiral modification occurring at the first, and second internucleotide linkages at the 5′ end of the antisense strand, having the linkage phosphorus atom in Rp configuration, and a terminal, chiral modification occurring at the first internucleotide linkage at the 5′ end of the sense strand, having the linkage phosphorus atom in either Rp or Sp configuration.

In one embodiment, the dsRNA agent further comprises a phosphate or phosphate mimic at the 5′-end of the antisense strand.

In one embodiment, the phosphate mimic is a 5′-vinyl phosphonate (VP).

In one embodiment, the base pair at the 1 position of the 5′-end of the antisense strand of the duplex is an AU base pair.

In one embodiment, the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.

The present invention further provides cells, pharmaceutical compositions for inhibiting expression of a MUC5B gene, and pharmaceutical composition comprising a lipid formulation. comprising the dsRNA agent of the invention.

In one aspect, the present invention provides a method of inhibiting expression of a MUC5B gene in a cell. The method includes contacting the cell with the dsRNA agent of the invention, or the pharmaceutical composition of the invention; and maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of a MUC5B gene, thereby inhibiting expression of the MUC5B gene in the cell.

In one embodiment, the cell is within a subject.

In one embodiment, the subject is a human.

In one embodiment, the expression of the MUC5B gene is inhibited by at least 50%.

In one aspect, the present invention provides a method of treating a MUC5B-associate disorder, e.g., a subject having a lung disease, e.g., pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF), cystic fibrosis, and/or chronic obstructive pulmonary disease (COPD), or a subject at risk of developing a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis, e.g., a subject carrying the carry the rs35705950 variant. The method includes administering to the subject a therapeutically effective amount of the dsRNA agent of the invention, or the pharmaceutical composition of the invention, thereby treating the subject.

In one embodiment, the subject is a human.

In one embodiment, treating comprises amelioration of at least on sign or symptom of the disease.

In one embodiment, the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg.

In one embodiment, the administration of the dsRNA is pulmonary system administration.

In some embodiments, the double stranded RNAi agent is administered to the subject intranasally, intratracheally, or by inhalation through the mouth. Certain devices are designed for delivery simultaneously through the mouth and nose. In some embodiments, the RNAi agent is administered to promote deposition substantially in the nasal cavity. In some embodiments, the RNAi agent is administered to promote deposition substantially in the lungs. In some embodiments, the RNAi agent is administered to promote deposition in the mouth or throat. In some embodiments, the RNAi agent is administered to promote deposition in both the nasal cavity and the lungs.

In certain embodiments, the RNAi agent is taken up in one or more tissues or cell types in the respiratory system including, but not limited to, bronchus, bronchiole, alveoli, epithelium including nasal and respiratory epithelium, ciliated epitheilium, and goblet cells; pneumocytes, both type I and type II, macrophages, peritubular interstitium, macrophages, adipose tissue, e.g., mediastinal adipose tissue, pulmonary neuronal cells, e.g., in the pulmonary neuroal plexus, club cells, clara cells, neutrophils, both resident and transient, and oral mucosa.

In certain embodiments, the RNAi agent is taken up on one or more tissue or cell types outside of the respiratory system, e.g., liver, kidney.

In one embodiment, the pulmonary system administration is oral inhalation or intranasally.

In one embodiment, the method reduces the expression of an MUC5B gene in a pulmonary system tissue, e.g., a nasopharynx tissue, an oropharynx tissue, a laryngopharynx tissue, a larynx tissue, a trachea tissue, a carina tissue, a bronchi tissue, a bronchiole tissue, or an alveoli tissue.

In one embodiment, the dsRNA agent is administered to the subject subcutaneously.

In one embodiment, the method further comprises administering to the subject an additional agent or a therapy suitable for treatment or prevention of a MUC5B-associated disorder.

In one embodiment, the additional therapeutic agent is selected from the group consisting of an anti-inflammatory agents (e.g., a systemic corticosteroid (e.g., prednisone), an immune modulator (e.g., an immunosuppressant agents (e.g., azathioprine, cyclophosphamide), a phosphodiesterase-5 inhibitor, a tyrosine kinase inhibitor (e.g., nintedanib), an antifibrotic agent (e.g., pirfenidone), and a combination of any of the foregoing.

The present invention is further illustrated by the following detailed description.

DETAILED DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph depicting mouse MUC5B mRNA levels COS7 cells 24 hours after transfection with the indicated duplexes at 10 nM, 1.0 nM, 0.1 nM. The level of mRNA is shown relative to the level of mouse MUC5B in COS7 cell transfected with a non-targeting siRNA.

FIG. 2 is a graph depicting the level of mouse MUC5B mRNA at Day 10 post-dose in whole lung lysates from mice administered a single 10 mg/kg dose of AD-1318337, AD-1318338, AD-1314054. AD-1317692, or AD-1318239, or saline control by orotracheal application on Day 0.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of a MUC5B gene. The MUC5B gene may be within a cell, e.g., a cell within a subject, such as a human. The use of these iRNAs enables the targeted degradation of mRNAs of the corresponding gene (a MUC5B gene) in mammals. The present disclosure also provides methods of using the RNAi compositions of the disclosure for inhibiting the expression of a MUC5B gene for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of a MUC5B gene, e.g., a MUC5B-associated disorder, e.g., a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF), e.g., a subject having IPF, or a subject at risk of IPF, e.g., a subject carrying the rs35705950 variant.

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is up to about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a MUC5B gene. In certain embodiments, the RNAi agents of the disclosure include an RNA strand (the antisense strand) having a region which is about 21-23 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of a MUC5B gene.

In certain embodiments, one or both of the strands of the double stranded RNAi agents of the invention is up to 66 nucleotides in length, e.g., 36-66, 26-36, 25-36, 31-60, 22-43, 27-53 nucleotides in length, with a region of at least 19 contiguous nucleotides that is substantially complementary to at least a part of an mRNA of a MUC5B gene. In some embodiments, such iRNA agents having longer length antisense strands preferably may include a second RNA strand (the sense strand) of 20-60 nucleotides in length wherein the sense and antisense strands form a duplex of 18-30 contiguous nucleotides.

The use of iRNAs of the invention enables the targeted degradation of the MUC5B mRNAs in mammals Thus, methods and compositions including these iRNAs are useful for treating a subject having a MUC5B-associated disorder, e.g., IPF, e.g., a subject having IPF, e.g., or treating a subject at risk of a IPF, e.g., a subject carrying the rs35705950 variant.

In certain embodiments, the administration of the dsRNA to a subject results in an improvement in lung function, or a stoppage or reduction of the rate of loss of lung function, or survival.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of a MUC5B gene s as well as compositions, uses, and methods for treating subjects that would benefit from inhibition and/or reduction of the expression of a MUC5B gene, e.g., subjects susceptible to or diagnosed with a MUC5B-associated disorder.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”.

The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges of tolerances in the art. For example, “about” can be understood as about 2 standard deviations from the mean. In certain embodiments, about means±10%. In certain embodiments, about means±5%. When about is present before a series of numbers or a range, it is understood that “about” can modify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understood to include the number adjacent to the term “at least”, and all subsequent numbers or integers that could logically be included, as clear from context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, “at least 18 nucleotides of a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series of numbers or a range, it is understood that “at least” can modify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex with an overhang of “no more than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “no more than” is present before a series of numbers or a range, it is understood that “no more than” can modify each of the numbers in the series or range. As used herein, ranges include both the upper and lower limit.

As used herein, methods of detection can include determination that the amount of analyte present is below the level of detection of the method.

In the event of a conflict between an indicated target site and the nucleotide sequence for a sense or antisense strand, the indicated sequence takes precedence.

In the event of a conflict between a sequence and its indicated site on a transcript or other sequence, the nucleotide sequence recited in the specification takes precedence.

As used herein, the term “Mucin 5B” (“MUC5B”) refers to the well-known gene and polypeptide, also known in the art as also referred to as “Mucin 5B, Oligomeric Mucus/Gel-Forming,” “High Molecular Weight Salivary Mucin MG1,” “Mucin 5, Subtype B, Tracheobronchial,” “Sublingual Gland Mucin,” “Mucin-5B,” “MUC-5B,” “MUC5,” “MG1,” “Mucin-5 Subtype B, Tracheobronchial,” “Cervical Mucin MUC5B,” “Cervical Mucin,” or “MUC9.” The term “MUC5B” includes human MUC5B, the amino acid and nucleotide sequences of which may be found in, for example, GenBank Accession No. NM_002458.3 (GI: 1519244536; SEQ ID NO:1); mouse MUC5B, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. NM_028801.2 (GI: 147905739, SEQ ID NO: 2); and rat MUC5B, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No.: XM_006230608.2 (GI: 672039062; SEQ ID NO: 3).

The term “MUC5B” also includes Macaca mulatta MUC5B, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. XM_028833012.1 (GI: 1622861542; SEQ ID NO:4) and Macaca fascicularis MUC5B, the amino acid and nucleotide sequence of which may be found in, for example, GenBank Accession No. XM_015435240.1 (GI: 982295518; SEQ ID NO:5).

Additional examples of MUC5B mRNA sequences are readily available using, e.g., GenBank, UniProt, OMIM, and the Macaca genome project web site.

Exemplary MUC5B nucleotide sequences may also be found in SEQ ID NOs:1-10. SEQ ID NOs:6-10 are the reverse complement sequences of SEQ ID NOs:1-5, respectively.

Further information on MUC5B is provided, for example in the NCBI Gene database at https://www.ncbi.nlm nih.gov/gene/727897.

The entire contents of each of the foregoing GenBank Accession numbers and the Gene database numbers are incorporated herein by reference as of the date of filing this application.

The terms “Mucin 5B” and “MUC5B,” as used herein, also refers to naturally occurring DNA sequence variations of the MUC5B gene. Numerous sequence variations within the MUC5B gene have been identified and may be found at, for example, NCBI dbSNP and UniProt (see, e.g., https://www.ncbi.nlm nih.gov/snp/?term=muc5b), the entire contents of which is incorporated herein by reference as of the date of filing this application.

Th term “rs35705950 variant” refers to the well-known promotor variant of a MUC5B gene significantly associated with both familial and sporadic idiopathic pulmonary fibrosis (IPF) and with increased MUC5B expression in lung tissue of unaffected subjects. Heterozygosity for rs35705950 (GT) is associated with a 6-fold increase in IPF and homozygosity (TT) is associated with a 20-fold increase in IPF.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a MUC5B gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for RNAi-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of a MUC5B gene. In one embodiment, the target sequence is within the protein coding region of the MUC5B gene. In another embodiment, the target sequence is within the 3′ UTR of the MUC5B gene.

The target sequence may be from about 9-36 nucleotides in length, e.g., preferably about 15-30 nucleotides in length. For example, the target sequence can be about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. In some embodiments, the target sequence is about 19 to about 30 nucleotides in length. In other embodiments, the target sequence is about 19 to about 25 nucleotides in length. In still other embodiments, the target sequence is about 19 to about 23 nucleotides in length. In some embodiments, the target sequence is about 21 to about 23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T,” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine, and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety (see, e.g., Table 1). The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. It is understood that when a cDNA sequence is provided, the corresponding mRNA or RNAi agent would include a U in place of a T. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention. Further, one of skill in the art that a T is a target gene sequence, or reverse complement thereof, would often be replaced by a U in an RNAi agent of the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,” “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. RNA interference (RNAi) is a process that directs the sequence-specific degradation of mRNA. RNAi modulates, e.g., inhibits, the expression of a MUC5B gene in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the disclosure includes a single stranded RNAi that interacts with a target RNA sequence, e.g., a MUC5B mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory it is believed that long double stranded RNA introduced into cells is broken down into double-stranded short interfering RNAs (siRNAs) comprising a sense strand and an antisense strand by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes these dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). These siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the disclosure relates to a single stranded RNA (ssRNA) (the antisense strand of a siRNA duplex) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA that is introduced into a cell or organism to inhibit a target mRNA. Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2, which then cleaves the target mRNA. The single-stranded siRNAs are generally 15-30 nucleotides and are chemically modified. The design and testing of single-stranded RNAs are described in U.S. Pat. No. 8,101,348 and in Lima et al., (2012) Cell 150:883-894, the entire contents of each of which are hereby incorporated herein by reference. Any of the antisense nucleotide sequences described herein may be used as a single-stranded siRNA as described herein or as chemically modified by the methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, a “RNAi agent” for use in the compositions and methods of the disclosure is a double stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., a MUC5B mRNA sequence. In some embodiments of the disclosure, a double stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, a dsRNA molecule can include ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide, a modified nucleotide. In addition, as used in this specification, an “RNAi agent” may include ribonucleotides with chemical modifications; an RNAi agent may include substantial modifications at multiple nucleotides.

As used herein, the term “modified nucleotide” refers to a nucleotide having, independently, a modified sugar moiety, a modified internucleotide linkage, or a modified nucleobase. Thus, the term modified nucleotide encompasses substitutions, additions or removal of, e.g., a functional group or atom, to internucleoside linkages, sugar moieties, or nucleobases. The modifications suitable for use in the agents of the disclosure include all types of modifications disclosed herein or known in the art. Any such modifications, as used in a siRNA type molecule, are encompassed by “RNAi agent” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of a deoxy-nucleotide—which is acknowledged as a naturally occurring form of nucleotide—if present within a RNAi agent can be considered to constitute a modified nucleotide.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides or nucleotides not directed to the target site of the dsRNA. In some embodiments, the hairpin loop can be or fewer nucleotides. In some embodiments, the hairpin loop can be 8 or fewer unpaired nucleotides. In some embodiments, the hairpin loop can be 4-10 unpaired nucleotides. In some embodiments, the hairpin loop can be 4-8 nucleotides.

In certain embodiment, the two strands of double-stranded oligomeric compound can be linked together. The two strands can be linked to each other at both ends, or at one end only. By linking at one end is meant that 5′-end of first strand is linked to the 3′-end of the second strand or 3′-end of first strand is linked to 5′-end of the second strand. When the two strands are linked to each other at both ends, 5′-end of first strand is linked to 3′-end of second strand and 3′-end of first strand is linked to 5′-end of second strand. The two strands can be linked together by an oligonucleotide linker including, but not limited to, (N)n; wherein N is independently a modified or unmodified nucleotide and n is 3-23. In some embodiments, n is 3-10, e.g., 3, 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the oligonucleotide linker is selected from the group consisting of GNRA, (G)4, (U)4, and (dT)4, wherein N is a modified or unmodified nucleotide and R is a modified or unmodified purine nucleotide. Some of the nucleotides in the linker can be involved in base-pair interactions with other nucleotides in the linker. The two strands can also be linked together by a non-nucleosidic linker, e.g. a linker described herein. It will be appreciated by one of skill in the art that any oligonucleotide chemical modifications or variations describe herein can be used in the oligonucleotide linker.

Hairpin and dumbbell type oligomeric compounds will have a duplex region equal to or at least 14, 15, 15, 16, 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region can be equal to or less than 200, 100, or 50, in length. In some embodiments, ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length.

The hairpin oligomeric compounds can have a single strand overhang or terminal unpaired region, in some embodiments at the 3′, and in some embodiments on the antisense side of the hairpin. In some embodiments, the overhangs are 1-4, more generally 2-3 nucleotides in length. The hairpin oligomeric compounds that can induce RNA interference are also referred to as “shRNA” herein.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

In one embodiment, an RNAi agent of the invention is a dsRNA, each strand of which is 24-30 nucleotides in length, that interacts with a target RNA sequence, e.g., a MUC5B mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188).

In one embodiment, an RNAi agent of the invention is a dsRNA agent, each strand of which comprises 19-23 nucleotides that interacts with a MUC5B mRNA sequence to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). In one embodiment, an RNAi agent of the invention is a dsRNA of 24-30 nucleotides that interacts with a MUC5B mRNA sequence to direct the cleavage of the target RNA.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of a RNAi agent, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively, the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment of the dsRNA, at least one strand comprises a 3′ overhang of at least 1 nucleotide. In another embodiment, at least one strand comprises a 3′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In other embodiments, at least one strand of the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. In certain embodiments, at least one strand comprises a 5′ overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 nucleotides. In still other embodiments, both the 3′ and the 5′ end of one strand of the RNAi agent comprise an overhang of at least 1 nucleotide.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or the antisense strand, or both, can include extended lengths longer than 10 nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30 nucleotides, or 10-15 nucleotides in length. In certain embodiments, an extended overhang is on the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the sense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the sense strand of the duplex. In certain embodiments, an extended overhang is on the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 3′end of the antisense strand of the duplex. In certain embodiments, an extended overhang is present on the 5′end of the antisense strand of the duplex. In certain embodiments, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate. In certain embodiments, the overhang includes a self-complementary portion such that the overhang is capable of forming a hairpin structure that is stable under physiological conditions.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., a MUC5B mRNA sequence.

As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., a MUC5B nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- or 3′-terminus of the RNAi agent.

In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the antisense strand. In some embodiments, the antisense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the target mRNA, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the target mRNA. In some embodiments, the antisense strand double stranded RNA agent of the invention includes no more than 4 mismatches with the sense strand, e.g., the antisense strand includes 4, 3, 2, 1, or 0 mismatches with the sense strand. In some embodiments, a double stranded RNA agent of the invention includes a nucleotide mismatch in the sense strand. In some embodiments, the sense strand of the double stranded RNA agent of the invention includes no more than 4 mismatches with the antisense strand, e.g., the sense strand includes 4, 3, 2, 1, or 0 mismatches with the antisense strand. In some embodiments, the nucleotide mismatch is, for example, within 5, 4, 3 nucleotides from the 3′-end of the iRNA. In another embodiment, the nucleotide mismatch is, for example, in the 3′-terminal nucleotide of the iRNA agent. In some embodiments, the mismatch(s) is not in the seed region.

Thus, an RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, a RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a MUC5B gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a MUC5B gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a MUC5B gene is important, especially if the particular region of complementarity in a MUC5B gene is known to vary.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of a RNAi agent that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides.

As used herein, the term “cleavage region” refers to a region that is located immediately adjacent to the cleavage site. The cleavage site is the site on the target at which cleavage occurs. In some embodiments, the cleavage region comprises three bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage region comprises two bases on either end of, and immediately adjacent to, the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bound by nucleotides 10 and 11 of the antisense strand, and the cleavage region comprises nucleotides 11, 12 and 13.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within a RNAi agent, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of a RNAi agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) or target sequence refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest or target sequence (e.g., an mRNA encoding MUC5B). For example, a polynucleotide is complementary to at least a part of a MUC5B RNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding MUC5B.

Accordingly, in some embodiments, the antisense strand polynucleotides disclosed herein are fully complementary to the target MUC5B sequence.

In other embodiments, the antisense strand polynucleotides disclosed herein are substantially complementary to the target MUC5B sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 1-5 for MUC5B, or a fragment of SEQ ID Nos: 1-5, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein are substantially complementary to the target MUC5B sequence and comprise a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the sense strand nucleotide sequences in any one of Tables 2-7, 9, and 10, or a fragment of any one of the sense strand nucleotide sequences in any one of Tables 2-7, 9, and 10, such as about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the disclosure includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is the same as a target MUC5B sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to the equivalent region of the nucleotide sequence of SEQ ID NOs: 6-10, or a fragment of any one of SEQ ID NOs: 6-10, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

In some embodiments, an iRNA of the invention includes a sense strand that is substantially complementary to an antisense polynucleotide which, in turn, is complementary to a target MUC5B sequence, and wherein the sense strand polynucleotide comprises a contiguous nucleotide sequence which is at least about 80% complementary over its entire length to any one of the antisense strand nucleotide sequences in any one of any one of Tables 2-7, 9, and 10, or a fragment of any one of the antisense strand nucleotide sequences in any one of Tables 2-7, 9, and 10, such as about 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or about 99% complementary.

In some embodiments, the double-stranded region of a double-stranded iRNA agent is equal to or at least, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, 30 or more nucleotide pairs in length.

In some embodiments, the antisense strand of a double-stranded iRNA agent is equal to or at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In some embodiments, the sense strand of a double-stranded iRNA agent is equal to or at least 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 15 to 30 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 19 to 25 nucleotides in length.

In one embodiment, the sense and antisense strands of the double-stranded iRNA agent are each independently 21 to 23 nucleotides in length.

In one embodiment, the sense strand of the iRNA agent is 21-nucleotides in length, and the antisense strand is 23-nucleotides in length, wherein the strands form a double-stranded region of 21 consecutive base pairs having a 2-nucleotide long single stranded overhangs at the 3′-end.

In one aspect of the invention, an agent for use in the methods and compositions of the invention is a single-stranded antisense nucleic acid molecule that inhibits a target mRNA via an antisense inhibition mechanism. The single-stranded antisense RNA molecule is complementary to a sequence within the target mRNA. The single-stranded antisense oligonucleotides can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol Cancer Ther 1:347-355. The single-stranded antisense RNA molecule may be about 15 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from any one of the antisense sequences described herein.

In one embodiment, at least partial suppression of the expression of a MUC5B gene, is assessed by a reduction of the amount of MUC5B mRNA which can be isolated from or detected in a first cell or group of cells in which a MUC5B gene is transcribed and which has or have been treated such that the expression of a MUC5B gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)} \cdot 100}\%$

In one embodiment, inhibition of expression is determined by the dual luciferase method wherein the RNAi agent is present at 10 nM.

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the RNAi agent or contacting a cell in vivo with the RNAi agent. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, via inhalation, intranasal administration, or intratracheal administration, by injecting the RNAi agent into or near the tissue where the cell is located, e.g., the pulmonary system, or by injecting the RNAi agent into another area, or to the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain or be coupled to a ligand, e.g., a lipophilic moiety or moieties as described below and further detailed, e.g., in PCT Publication No. WO 2019/217459, the entire contents of which is incorporated herein by reference, that directs or otherwise stabilizes the RNAi agent at a site of interest, e.g., the pulmonary system. In some embodiments, the RNAi agent may contain or be coupled to a ligand, e.g., one or more GalNAc derivatives as described below, that directs or otherwise stabilizes the RNAi agent at a site of interest, e.g., the liver. In other embodiments, the RNAi agent may contain or be coupled to a lipophilic moiety or moieties and one or more GalNAc derivatives. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an RNAi agent includes “introducing” or “delivering the RNAi agent into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of a RNAi agent can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing a RNAi agent into a cell may be in vitro or in vivo. For example, for in vivo introduction, a RNAi agent can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below or are known in the art.

The term “lipophile” or “lipophilic moiety” broadly refers to any compound or chemical moiety having an affinity for lipids. One way to characterize the lipophilicity of the lipophilic moiety is by the octanol-water partition coefficient, log K_(ow), where K_(ow) is the ratio of a chemical's concentration in the octanol-phase to its concentration in the aqueous phase of a two-phase system at equilibrium. The octanol-water partition coefficient is a laboratory-measured property of a substance. However, it may also be predicted by using coefficients attributed to the structural components of a chemical which are calculated using first-principle or empirical methods (see, for example, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001), which is incorporated herein by reference in its entirety). It provides a thermodynamic measure of the tendency of the substance to prefer a non-aqueous or oily milieu rather than water (i.e. its hydrophilic/lipophilic balance). In principle, a chemical substance is lipophilic in character when its log K_(ow) exceeds 0. Typically, the lipophilic moiety possesses a log K_(ow) exceeding 1, exceeding 1.5, exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. For instance, the log K_(ow) of 6-amino hexanol, for instance, is predicted to be approximately 0.7. Using the same method, the log K_(ow) of cholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.

The lipophilicity of a molecule can change with respect to the functional group it carries. For instance, adding a hydroxyl group or amine group to the end of a lipophilic moiety can increase or decrease the partition coefficient (e.g., log K_(ow)) value of the lipophilic moiety.

Alternatively, the hydrophobicity of the double-stranded RNAi agent, conjugated to one or more lipophilic moieties, can be measured by its protein binding characteristics. For instance, in certain embodiments, the unbound fraction in the plasma protein binding assay of the double-stranded RNAi agent could be determined to positively correlate to the relative hydrophobicity of the double-stranded RNAi agent, which could then positively correlate to the silencing activity of the double-stranded RNAi agent.

In one embodiment, the plasma protein binding assay determined is an electrophoretic mobility shift assay (EMSA) using human serum albumin protein. An exemplary protocol of this binding assay is illustrated in detail in, e.g., PCT Publication No. WO 2019/217459. The hydrophobicity of the double-stranded RNAi agent, measured by fraction of unbound siRNA in the binding assay, exceeds 0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4, exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.

Accordingly, conjugating the lipophilic moieties to the internal position(s) of the double-stranded RNAi agent provides optimal hydrophobicity for the enhanced in vivo delivery of siRNA.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipid layer encapsulating a pharmaceutically active molecule, such as a nucleic acid molecule, e.g., an RNAi agent or a plasmid from which a RNAi agent is transcribed. LNPs are described in, for example, U.S. Pat. Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), or a non-primate (such as a a cow, a pig, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, or a mouse), or a bird that expresses the target gene, either endogenously or heterologously. In a preferred embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder, or condition that would benefit from reduction in MUC5B expression; a human at risk for a disease, disorder, or condition that would benefit from reduction in MUC5B expression; a human having a disease, disorder, or condition that would benefit from reduction in MUC5B expression; or human being treated for a disease, disorder, or condition that would benefit from reduction in MUC5B expression as described herein. In some embodiments, the subject is a female human. In other embodiments, the subject is a male human. In one embodiment, the subject is an adult subject. In another embodiment, the subject is a pediatric subject.

As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, but not limited to, alleviation or amelioration of one or more signs or symptoms associated with MUC5B expression or MUC5B protein production, e.g., a MUC5B-associated disease, e.g., a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF) or symptoms associated with unwanted MUC5B expression; diminishing the extent of unwanted MUC5B activation or stabilization; amelioration or palliation of unwanted MUC5B activation or stabilization. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

The term “lower” in the context of the level of MUC5B in a subject or a disease marker or symptom refers to a statistically significant decrease in such level. The decrease can be, for example, at least 10%, 15%, 20%, 25%, 30%, %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more. In certain embodiments, a decrease is at least 20%. In certain embodiments, the decrease is at least 50% in a disease marker, e.g., protein or gene expression level. “Lower” in the context of the level of MUC5B in a subject is preferably down to a level accepted as within the range of normal for an individual without such disorder. In certain embodiments, the expression of the target is normalized, i.e., decreased towards or to a level accepted as within the range of normal for an individual without such disorder, e.g., blood oxygen level, white blood cell count, kidney function, liver function. As used here, “lower” in a subject can refer to lowering of gene expression or protein production in a cell in a subject does not require lowering of expression in all cells or tissues of a subject. For example, as used herein, lowering in a subject can include lowering of gene expression or protein production in a subject.

The term “lower” can also be used in association with normalizing a symptom of a disease or condition, i.e. decreasing the difference between a level in a subject suffering from a MUC5B-associated disease towards or to a level in a normal subject not suffering from a MUC5B-associated disease. As used herein, if a disease is associated with an elevated value for a symptom, “normal” is considered to be the upper limit of normal. If a disease is associated with a decreased value for a symptom, “normal” is considered to be the lower limit of normal.

As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder, or condition thereof, that would benefit from a reduction in expression of a MUC5B gene or production of a MUC5B protein, refers to a reduction in the likelihood that a subject will develop a symptom associated with such a disease, disorder, or condition, e.g., a symptom of a MUC5B-associated disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., IPF. The failure to develop a disease, disorder, or condition, or the reduction in the development of a symptom associated with such a disease, disorder, or condition (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “MUC5B-associated disease,” is a disease or disorder that would benefit from reduction in the expression or activity of MUC5B. Such MUC5B-associated diseases include a MUC5B-associated disease.

The term “MUC5B-associated disease,” is a disease or disorder that is caused by, or associated with MUC5B expression or MUC5B protein production. The term “MUC5B-associated disease” includes a disease, disorder or condition that would benefit from a decrease in MUC5B expression or MUC5B protein activity. Non-limiting examples of MUC5B-associated diseases include, for example, lung diseases, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF).

As used herein, the term “pulmonary fibrosis” refers to a condition of the lungs in which the tissue thickens and becomes scarred. This thickened, stiff tissue makes it more difficult for the lungs to work properly. As pulmonary fibrosis worsens, people become progressively more short of breath. In some embodiments, the cause of pulmonary fibrosis is unknown. In those instances, the pulmonary fibrosis is referred to as “idiopathic pulmonary fibrosis (IPF)”.

As used herein, the term “chronic obstructive pulmonary disease (COPD)” refers to a disease of the lung characterized by chronic obstruction of airflow. In COPD, the damage accrued by the lungs over time leads to a loss in elasticity of the lung tissue that is responsible for proper exhalation. When this elasticity is lost, some waste carbon dioxide is left in the lungs at the end of exhalation, leading to carbon dioxide buildup in the body. COPD leads to emphysema, which is the destruction of the alveoli, and chronic bronchitis, which is inflammation of the airway tubes in the lungs.

As used herein, the term “cystic fibrosis” refers to a genetic disorder that results in thickening tissue and buildup of mucus in the lungs, pancreas, liver, kidneys and intestines. Individuals with cystic fibrosis develop a thick mucus that can block the airways in the lungs. This mucus buildup results in troubled breathing and an increased susceptibility to respiratory infections, as mucus traps the bacteria and is unable to be removed efficiently. This condition also has severely debilitating effects on the digestive system, resulting in stunted growth and weight.

The symptoms for a MUC5B-associated disease include, for example, exertional dyspnea, a nonproductive cough, weight loss, low-grade fevers, fatigue, arthralgias, fine bibasilar inspiratory crackles (Velcro crackles), digital clubbing, pulmonary hypertension at rest, loud P2 component of the second heart sound, a fixed split S2, a holosystolic tricuspid regurgitation murmur, pedal edema, histopathologic and/or radiologic pattern of usual interstitial pneumonia (UIP), mucus buildup in the airways, troubled breathing, an increased susceptibility to respiratory infections, stunted growth and weight, a loss in elasticity of the lung tissue, carbon dioxide buildup in the body, emphysema, chronic bronchitis, shortness of breath, a chronic cough and excessive mucus, wheezing, a tight feeling in the chest, blue lips and nail beds, and uncontrollable weight loss.

Further details regarding signs and symptoms of the various diseases or conditions are provided herein and are well known in the art.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a MUC5B-associated disease, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating, or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of a RNAi agent that, when administered to a subject having a MUC5B-associated disorder, e.g., a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, such as IPF, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the RNAi agent, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of a RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. A RNAi agent employed in the methods of the present disclosure may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. Pharmaceutically acceptable carriers for pulmonary delivery are known in the art and will vary depending on the desired location for deposition of the agent, e.g., upper or lower respiratory system, and the type of device to be used for delivery, e.g., sprayer, nebulizer, dry powder inhaler.

As used herein, “respiratory system” is understood as the structures through which air moves from outside the body into the lungs and back out, e.g., the mouth, nose and nasal cavity, sinus, trachea, pharynyx, larynx, bronchial tubes/bronchi, bronchioles, alveoli, and vasculature, e.g., capillaries, hematopoietic cells, lymphatics, and lungs, and the cells, tissues, and fluids present therein.

As used herein, “delivery by inhalation” and the like include delivery by inhalation through the nose or mouth, including intratracheal administration. Delivery by inhalation typically is performed using a device, e.g., inhaler, sprayer, nebulizer, that is selected, in part, based on the location that the agent is to be delivered, e.g., nose, mouth, lungs, and the type of material to be delivered, e. g., drops, mist, dry powder.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, bronchial fluids, sputum, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, sputum, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from a nasal swab. In certain embodiments, samples may be derived from a throat swab. In certain embodiments, samples may be derived from the lung, or certain types of cells in the lung. In some embodiments, the samples may be derived from the bronchioles. In some embodiments, the samples may be derived from the bronchus. In some embodiments, the samples may be derived from the alveoli. In other embodiments, a “sample derived from a subject” refers to liver tissue (or subcomponents thereof) derived from the subject. In some embodiments, a “sample derived from a subject” refers to blood drawn from the subject or plasma or serum derived therefrom. In further embodiments, a “sample derived from a subject” refers to pulmonary tissue (or subcomponents thereof) derived from the subject.

II. RNAi Agents of the Disclosure

Described herein are RNAi agents which inhibit the expression of a MUC5B gene. In one embodiment, the RNAi agent includes double stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of a MUC5B gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human, e.g., a subject having a MUC5B-associated disorder, e.g., a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., IPF, or a subject at risk of a MUC5B-associated disease, such as IPF, e.g., a subject carrying an rs35705950 variant.

The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of a target RNA, e.g., an mRNA formed in the expression of a MUC5B gene. The region of complementarity is about 15-30 nucleotides or less in length. Upon contact with a cell expressing the MUC5B gene, the RNAi agent inhibits the expression of the MUC5B gene (e.g., a human gene, a primate gene, a non-primate gene) by at least 50% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, western blotting or flow cytometric techniques. In preferred embodiments, inhibition of expression is by at least 50% as assayed by the Dual-Glo lucifierase assay in Example 1 where the siRNA is at a 10 nM concentration.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. For example, the target sequence can be derived from the sequence of an mRNA formed during the expression of a MUC5B gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is 15 to 30 base pairs in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. In certain preferred embodiments, the duplex structure is 18 to 25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24,20-23, 20-22, 20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or 24-25 base pairs in length, for example, 19-21 basepairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

Similarly, the region of complementarity to the target sequence is 15 to 30 nucleotides in length, e.g., 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, for example 19-23 nucleotides in length or 21-23 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the disclosure.

In some embodiments, the dsRNA is 15 to 23 nucleotides in length, or 25 to 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 15 to 36 base pairs, e.g., 15-36, 15-35, 15-34, 15-33, 15-32, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs, for example, 19-21 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, a RNAi agent useful to target MUC5B expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA. In certain embodiments, longer, extended overhangs are possible.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

An siRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.

An siRNA can be made by separately synthesizing a single stranded RNA molecule, or each respective strand of a double-stranded RNA molecule, after which the component strands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given siRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the siRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.

Organic synthesis can be used to produce a discrete siRNA species. The complementary of the species to a MUC5B gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.

In one embodiment, RNA generated is carefully purified to remove endsiRNA is cleaved in vitro into siRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsiRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g., a purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9 and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsiRNA cleavage generally produces a plurality of siRNA species, each being a particular 21 to 23 nucleotide fragment of a source dsiRNA molecule. For example, siRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsiRNA molecule may be present.

Regardless of the method of synthesis, the siRNA preparation can be prepared in a solution (e.g., an aqueous or organic solution) that is appropriate for formulation. For example, the siRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried siRNA can then be resuspended in a solution appropriate for the intended formulation process.

In one aspect, a dsRNA of the disclosure includes at least two nucleotide sequences, a sense sequence and an antisense sequence. The sense strand sequence for MUC5B may be selected from the group of sequences provided in any one of Tables 2-7, 9, and 10, and the corresponding nucleotide sequence of the antisense strand of the sense strand may be selected from the group of sequences of any one of Tables 2-7, 9, and 10. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of a MUC5B gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand (passenger strand) in any one of Tables 2-7, 9, and 10, and the second oligonucleotide is described as the corresponding antisense strand (guide strand) of the sense strand in any one of Tables 2-7, 9, and for MUC5B.

In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

It will be understood that, although the sequences provided herein are described as modified or conjugated sequences, the RNA of the RNAi agent of the disclosure e.g., a dsRNA of the disclosure, may comprise any one of the sequences set forth in any one of Tables 2-7, 9, and 10 that is un-modified, un-conjugated, or modified or conjugated differently than described therein. One or more lipophilic ligands or one or more GalNAc ligands can be included in any of the positions of the RNAi agents provided in the instant application.

The skilled person is well aware that dsRNAs having a duplex structure of about 20 to 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided herein, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences provided herein, and differing in their ability to inhibit the expression of a MUC5B gene by not more than 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence using the in vitro assay with Cos7 and a 10 nM concentration of the RNA agent and the PCR assay as provided in the examples herein, are contemplated to be within the scope of the present disclosure.

In addition, the RNAs described herein identify a site(s) in a MUC5B transcript that is susceptible to RISC-mediated cleavage. As such, the present disclosure further features RNAi agents that target within this site(s). As used herein, a RNAi agent is said to target within a particular site of an RNA transcript if the RNAi agent promotes cleavage of the transcript anywhere within that particular site. Such a RNAi agent will generally include at least about 15 contiguous nucleotides, preferably at least 19 nucleotides, from one of the sequences provided herein coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in a MUC5B gene.

An RNAi agent as described herein can contain one or more mismatches to the target sequence. In one embodiment, an RNAi agent as described herein contains no more than 3 mismatches (i.e., 3, 2, 1, or 0 mismatches). In one embodiment, an RNAi agent as described herein contains no more than 2 mismatches. In one embodiment, an RNAi agent as described herein contains no more than 1 mismatch. In one embodiment, an RNAi agent as described herein contains 0 mismatches. In certain embodiments, if the antisense strand of the RNAi agent contains mismatches to the target sequence, the mismatch can optionally be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, in such embodiments, for a 23 nucleotide RNAi agent, the strand which is complementary to a region of a MUC5B gene generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an RNAi agent containing a mismatch to a target sequence is effective in inhibiting the expression of a MUC5B gene. Consideration of the efficacy of RNAi agents with mismatches in inhibiting expression of a MUC5B gene is important, especially if the particular region of complementarity in a MUC5B gene is known to mutate.

III. Modified RNAi Agents of the Disclosure

In one embodiment, the RNA of the RNAi agent of the disclosure e.g., a dsRNA, is un-modified, and does not comprise, e.g., chemical modifications or conjugations known in the art and described herein. In preferred embodiments, the RNA of an RNAi agent of the disclosure, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. In certain embodiments of the disclosure, substantially all of the nucleotides of an RNAi agent of the disclosure are modified. In other embodiments of the disclosure, all of the nucleotides of an RNAi agent of the disclosure are modified. RNAi agents of the disclosure in which “substantially all of the nucleotides are modified” are largely but not wholly modified and can include not more than 5, 4, 3, 2, or 1 unmodified nucleotides. In still other embodiments of the disclosure, RNAi agents of the disclosure can include not more than 5, 4, 3, 2 or 1 modified nucleotides.

The nucleic acids featured in the disclosure can be synthesized or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of RNAi agents useful in the embodiments described herein include, but are not limited to, RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified RNAi agent will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, e.g., sodium salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in RNAi agents, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the RNAi agents of the disclosure are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the disclosure include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The RNAi agents, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)_(−n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a RNAi agent, or a group for improving the pharmacodynamic properties of a RNAi agent, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include: 5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides, 5′-Me-2′-deoxynucleotides, (both R and S isomers in these three families); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH 3), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-O-hexadecyl, and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of a RNAi agent, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. RNAi agents can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An RNAi agent of the disclosure can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the disclosure. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

An RNAi agent of the disclosure can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

An RNAi agent of the disclosure can also be modified to include one or more bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ring modified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”) is a nucleoside having a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4′-carbon and the 2′-carbon of the sugar ring. Thus, in some embodiments an agent of the disclosure may include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. In other words, an LNA is a nucleotide comprising a bicyclic sugar moiety comprising a 4′-CH2-O-2′ bridge. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the disclosure include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, the antisense polynucleotide agents of the disclosure include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2′; 4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrained ethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (and analogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′ (see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S. Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are hereby incorporated herein by reference.

Additional representative US patents and US patent Publications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133; 7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193; 8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US 2009/0012281, the entire contents of each of which are hereby incorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations including for example α-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

An RNAi agent of the disclosure can also be modified to include one or more constrained ethyl nucleotides. As used herein, a “constrained ethyl nucleotide” or “cEt” is a locked nucleic acid comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)-O-2′ bridge. In one embodiment, a constrained ethyl nucleotide is in the S conformation referred to herein as “S-cEt.”

An RNAi agent of the disclosure may also include one or more “conformationally restricted nucleotides” (“CRN”). CRN are nucleotide analogs with a linker connecting the C2′ and C4′ carbons of ribose or the —C3′ and —C5′ carbons of ribose. CRN lock the ribose ring into a stable conformation and increase the hybridization affinity to mRNA. The linker is of sufficient length to place the oxygen in an optimal position for stability and affinity resulting in less ribose ring puckering.

Representative publications that teach the preparation of certain of the above noted CRN include, but are not limited to, US 2013/0190383; and WO 2013/036868, the entire contents of each of which are hereby incorporated herein by reference.

In some embodiments, a RNAi agent of the disclosure comprises one or more monomers that are UNA (unlocked nucleic acid) nucleotides. UNA is unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomer with bonds between C1′-C4′ have been removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated by reference).

Representative U.S. publications that teach the preparation of UNA include, but are not limited to, U.S. Pat. No. 8,314,227; and US Patent Publication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in WO 2011/005861.

Other modifications of a RNAi agent of the disclosure include a 5′ phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate or phosphate mimic on the antisense strand of a RNAi agent. Suitable phosphate mimics are disclosed in, for example US 2012/0157511, the entire contents of which are incorporated herein by reference.

A. Modified RNAi Agents Comprising Motifs of the Disclosure

In certain aspects of the disclosure, the double-stranded RNAi agents of the disclosure include agents with chemical modifications as disclosed, for example, in WO 2013/075035, the entire contents of which are incorporated herein by reference. As shown herein and in WO 2013/075035, a superior result may be obtained by introducing one or more motifs of three identical modifications on three consecutive nucleotides into a sense strand or antisense strand of an RNAi agent, particularly at or near the cleavage site. In some embodiments, the sense strand and antisense strand of the RNAi agent may otherwise be completely modified. The introduction of these motifs interrupts the modification pattern, if present, of the sense or antisense strand. The RNAi agent may be optionally conjugated with a lipophilic ligand, e.g., a C16 ligand, for instance on the sense strand. The RNAi agent may be optionally modified with a (S)-glycol nucleic acid (GNA) modification, for instance on one or more residues of the antisense strand. The resulting RNAi agents present superior gene silencing activity.

Accordingly, the disclosure provides double stranded RNAi agents capable of inhibiting the expression of a targetgenome or gene (i.e., a MUC5B gene) in vivo. The RNAi agent comprises a sense strand and an antisense strand. Each strand of the RNAi agent may be 15-30 nucleotides in length. For example, each strand may be 16-30 nucleotides in length, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30 nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides in length, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23 nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides in length, or 21-23 nucleotides in length. In certain embodiments, each strand is 19-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex double stranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” The duplex region of an RNAi agent may be 15-30 nucleotide pairs in length. For example, the duplex region can be 16-30 nucleotide pairs in length, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs in length, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs in length, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs in length, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs in length, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs in length. In another example, the duplex region is selected from 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length. In preferred embodiments, the duplex region is 19-21 nucleotide pairs in length.

In one embodiment, the RNAi agent may contain one or more overhang regions or capping groups at the 3′-end, 5′-end, or both ends of one or both strands. The overhang can be 1-6 nucleotides in length, for instance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5 nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides in length, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2 nucleotides in length. In preferred embodiments, the nucleotide overhang region is 2 nucleotides in length. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence. The first and second strands can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAi agent can each independently be a modified or unmodified nucleotide including, but no limited to 2′-sugar modified, such as, 2-F, 2′-O-methyl, thymidine (T), and any combinations thereof.

For example, TT can be an overhang sequence for either end on either strand. The overhang can form a mismatch with the target mRNA or it can be complementary to the gene sequences being targeted or can be another sequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or both strands of the RNAi agent may be phosphorylated. In some embodiments, the overhang region(s) contains two nucleotides having a phosphorothioate between the two nucleotides, where the two nucleotides can be the same or different. In one embodiment, the overhang is present at the 3′-end of the sense strand, antisense strand, or both strands. In one embodiment, this 3′-overhang is present in the antisense strand. In one embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthen the interference activity of the RNAi, without affecting its overall stability. For example, the single-stranded overhang may be located at the 3′-terminal end of the sense strand or, alternatively, at the 3′-terminal end of the antisense strand. The RNAi may also have a blunt end, located at the 5′-end of the antisense strand (or the 3′-end of the sense strand) or vice versa. Generally, the antisense strand of the RNAi has a nucleotide overhang at the 3′-end, and the 5′-end is blunt. While not wishing to be bound by theory, the asymmetric blunt end at the 5′-end of the antisense strand and 3′-end overhang of the antisense strand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 7, 8, 9 from the 5′end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 8, 9, 10 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of 21 nucleotides in length, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strand and a 23 nucleotide antisense strand, wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides at positions 9, 10, 11 from the 5′ end; the antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at positions 11, 12, 13 from the 5′end, wherein one end of the RNAi agent is blunt, while the other end comprises a 2 nucleotide overhang. Preferably, the 2 nucleotide overhang is at the 3′-end of the antisense strand. When the 2 nucleotide overhang is at the 3′-end of the antisense strand, there may be two phosphorothioate internucleotide linkages between the terminal three nucleotides, wherein two of the three nucleotides are the overhang nucleotides, and the third nucleotide is a paired nucleotide next to the overhang nucleotide. In one embodiment, the RNAi agent additionally has two phosphorothioate internucleotide linkages between the terminal three nucleotides at both the 5′-end of the sense strand and at the 5′-end of the antisense strand. In one embodiment, every nucleotide in the sense strand and the antisense strand of the RNAi agent, including the nucleotides that are part of the motifs are modified nucleotides. In one embodiment each residue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g., in an alternating motif. Optionally, the RNAi agent further comprises a ligand (e.g., a lipophilic ligand, optionally a C16 ligand).

In one embodiment, the RNAi agent comprises a sense and an antisense strand, wherein the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1) positions 1 to 23 of the first strand comprise at least 8 ribonucleotides; the antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, comprises at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3 ‘ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3’ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when the double stranded nucleic acid is introduced into a mammalian cell; and wherein the sense strand contains at least one motif of three 2′-F modifications on three consecutive nucleotides, where at least one of the motifs occurs at or near the cleavage site. The antisense strand contains at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands, wherein the RNAi agent comprises a first strand having a length which is at least 25 and at most 29 nucleotides and a second strand having a length which is at most 30 nucleotides with at least one motif of three 2′-O-methyl modifications on three consecutive nucleotides at position 11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand and the 5′ end of the second strand form a blunt end and the second strand is 1˜4 nucleotides longer at its 3′ end than the first strand, wherein the duplex region region which is at least 25 nucleotides in length, and the second strand is sufficiently complemenatary to a target mRNA along at least 19 nucleotide of the second strand length to reduce target gene expression when the RNAi agent is introduced into a mammalian cell, and wherein dicer cleavage of the RNAi agent preferentially results in an siRNA comprising the 3′ end of the second strand, thereby reducing expression of the target gene in the mammal. Optionally, the RNAi agent further comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at the cleavage site in the sense strand.

In one embodiment, the antisense strand of the RNAi agent can also contain at least one motif of three identical modifications on three consecutive nucleotides, where one of the motifs occurs at or near the cleavage site in the antisense strand.

For an RNAi agent having a duplex region of 17-23 nucleotide in length, the cleavage site of the antisense strand is typically around the 10, 11 and 12 positions from the 5′-end. Thus the motifs of three identical modifications may occur at the 9, 10, 11 positions; 10, 11, 12 positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15 positions of the antisense strand, the count starting from the 1″ nucleotide from the 5′-end of the antisense strand, or, the count starting from the 1″ paired nucleotide within the duplex region from the 5′-end of the antisense strand. The cleavage site in the antisense strand may also change according to the length of the duplex region of the RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif of three identical modifications on three consecutive nucleotides at the cleavage site of the strand; and the antisense strand may have at least one motif of three identical modifications on three consecutive nucleotides at or near the cleavage site of the strand. When the sense strand and the antisense strand form a dsRNA duplex, the sense strand and the antisense strand can be so aligned that one motif of the three nucleotides on the sense strand and one motif of the three nucleotides on the antisense strand have at least one nucleotide overlap, i.e., at least one of the three nucleotides of the motif in the sense strand forms a base pair with at least one of the three nucleotides of the motif in the antisense strand. Alternatively, at least two nucleotides may overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides. The first motif may occur at or near the cleavage site of the strand and the other motifs may be a wing modification. The term “wing modification” herein refers to a motif occurring at another portion of the strand that is separated from the motif at or near the cleavage site of the same strand. The wing modification is either adjacent to the first motif or is separated by at least one or more nucleotides. When the motifs are immediately adjacent to each other then the chemistry of the motifs are distinct from each other and when the motifs are separated by one or more nucleotide than the chemistries can be the same or different. Two or more wing modifications may be present. For instance, when two wing modifications are present, each wing modification may occur at one end relative to the first motif which is at or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent may contain more than one motif of three identical modifications on three consecutive nucleotides, with at least one of the motifs occurring at or near the cleavage site of the strand. This antisense strand may also contain one or more wing modifications in an alignment similar to the wing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two terminal nucleotides at the 3′-end, 5′-end or both ends of the strand.

In another embodiment, the wing modification on the sense strand or antisense strand of the RNAi agent typically does not include the first one or two paired nucleotides within the duplex region at the 3′-end, 5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent each contain at least one wing modification, the wing modifications may fall on the same end of the duplex region, and have an overlap of one, two or three nucleotides.

When the sense strand and the antisense strand of the RNAi agent each contain at least two wing modifications, the sense strand and the antisense strand can be so aligned that two modifications each from one strand fall on one end of the duplex region, having an overlap of one, two or three nucleotides; two modifications each from one strand fall on the other end of the duplex region, having an overlap of one, two or three nucleotides; two modifications one strand fall on each side of the lead motif, having an overlap of one, two, or three nucleotides in the duplex region.

In one embodiment, the RNAi agent comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch may occur in the overhang region or the duplex region. The base pair may be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand independently selected from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strand is deoxy-thymine (dT). In another embodiment, the nucleotide at the 3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment, there is a short sequence of deoxy-thymine nucleotides, for example, two dT nucleotides on the 3′-end of the sense or antisense strand.

In one embodiment, the sense strand sequence may be represented by formula (I):

5′n _(p)-N_(a)—(X X X)_(i)-N_(b)-Y Y Y-N_(b)—(Z Z Z)_(j)N_(a)-n _(q)3′  (I)

-   -   wherein:     -   i and j are each independently 0 or 1;     -   p and q are each independently 0-6;     -   each N_(a) independently represents an oligonucleotide sequence         comprising 0-25 modified nucleotides, each sequence comprising         at least two differently modified nucleotides;     -   each N_(b) independently represents an oligonucleotide sequence         comprising 0-10 modified nucleotides;     -   each n p and n q independently represent an overhang nucleotide;     -   wherein N_(b) and Y do not have the same modification; and     -   XXX, YYY and ZZZ each independently represent one motif of three         identical modifications on three consecutive nucleotides.         Preferably YYY is all 2′-F modified nucleotides.

In one embodiment, the N_(a) or N_(b) comprise modifications of alternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site of the sense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotides in length, the YYY motif can occur at or the vicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7, 8, 9, 8, 9, 10, 9, 11, 10, 11,12 or 11, 12, 13) of—the sense strand, the count starting from the 1″ nucleotide, from the 5′-end; or optionally, the count starting at the 1″ paired nucleotide within the duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both i and j are 1. The sense strand can therefore be represented by the following formulas:

5′n _(p)-N_(a)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′  (Ib);

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(a)-n _(q)3′  (Ic); or

5′n _(p)-N_(a)-XXX-N_(b)-YYY-N_(b)-ZZZ-N_(a)-n _(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides.

Each N a independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N a can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Id), each N_(b) independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6. Each N a can independently represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may be represented by the formula:

5′n _(p)-N_(a)-YYY-N_(a)-n _(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N a independently can represent an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may be represented by formula (II):

5′n _(q′)-N_(a)′-(Z′Z′Z′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(X′X′X′)_(l)-N′_(a)-n _(p)′3′  (II)

-   -   wherein:     -   k and l are each independently 0 or 1;     -   p′ and q′ are each independently 0-6;         each N_(a)′ independently represents an oligonucleotide sequence         comprising 0-25 modified nucleotides, each sequence comprising         at least two differently modified nucleotides;         each N_(b)′ independently represents an oligonucleotide sequence         comprising 0-10 modified nucleotides;         each n_(p)′ and n_(q)′ independently represent an overhang         nucleotide;         wherein N_(b)′ and Y′ do not have the same modification; and         X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif         of three identical modifications on three consecutive         nucleotides.

In one embodiment, the N_(a)′ or N_(b)′ comprise modifications of alternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisense strand. For example, when the RNAi agent has a duplex region of 17-23 nucleotide in length, the Y′Y′Y′ motif can occur at positions 9, 10, 11; 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisense strand, with the count starting from the 1″ nucleotide, from the 5′-end; or optionally, the count starting at the 1″ paired nucleotide within the duplex region, from the 5′-end. Preferably, the Y′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and 1 is 0, or k is 0 and l is 1, or both k and l are 1.

The antisense strand can therefore be represented by the following formulas:

5′n _(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(a)′-n _(p′)3′  (IIb);

5′n _(q′)-N_(a)′-Y′Y′Y′-N_(b)′-X′X′X′-n _(p′)3′  (IIc); or

5′n _(q′)-N_(a)′-Z′Z′Z′-N_(b)′-Y′Y′Y′-N_(b)′-X′X′X′-N_(a)′-n _(p′)3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IIe), N_(b)′ represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4, 5 or 6.

In other embodiments, k is 0 and l is 0 and the antisense strand may be represented by the formula:

5′n _(p′)-N_(a)′-Y′Y′Y′-N_(a′)-n _(q′)3′  (Ia).

When the antisense strand is represented as formula (Ha), each N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may be independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. For example, each nucleotide of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′, Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a 2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYY motif occurring at 9, 10 and 11 positions of the strand when the duplex region is 21 nt, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y represents 2′-F modification. The sense strand may additionally contain XXX motif or ZZZ motifs as wing modifications at the opposite end of the duplex region; and XXX and ZZZ each independently represents a 2′-OMe modification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motif occurring at positions 11, 12, 13 of the strand, the count starting from the 1^(st) nucleotide from the 5′-end, or optionally, the count starting at the 1^(st) paired nucleotide within the duplex region, from the 5′-end; and Y′ represents 2′-O-methyl modification. The antisense strand may additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wing modifications at the opposite end of the duplex region; and X′X′X′ and Z′Z′Z′ each independently represents a 2′-OMe modification or 2′-F modification.

The sense strand represented by any one of the above formulas (Ia), (Ib), (Ic), and (Id) forms a duplex with a antisense strand being represented by any one of formulas (IIa), (IIb), (IIc), and (IId), respectively.

Accordingly, the RNAi agents for use in the methods of the disclosure may comprise a sense strand and an antisense strand, each strand having 14 to 30 nucleotides, the RNAi duplex represented by formula (III):

sense: 5′n _(p)-N_(a)-(X X X)_(i)-N_(b)-Y Y Y-N_(b)-(Z Z Z)_(j)-N_(a)-n _(q)3′

antisense: 3′n _(p)′-N_(a)′-(X′X′X′)_(k)-N_(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N_(a)′-n _(q)′5′   (III)

-   -   wherein:     -   j, k, and l are each independently 0 or 1;     -   p, p′, q, and q′ are each independently 0-6;     -   each N_(a) and N_(a)′ independently represents an         oligonucleotide sequence comprising 0-25 modified nucleotides,         each sequence comprising at least two differently modified         nucleotides;     -   each N_(b) and N_(b)′ independently represents an         oligonucleotide sequence comprising 0-10 modified nucleotides;     -   wherein     -   each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may         not be present, independently represents an overhang nucleotide;         and     -   XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently         represent one motif of three identical modifications on three         consecutive nucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0 and j is 1; or both i and j are 0; or both i and j are 1. In another embodiment, k is 0 and l is 0; or k is 1 and l is 0; k is 0 and l is 1; or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forming a RNAi duplex include the formulas below:

5′n _(p)-N_(a)-Y Y Y-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-Y′Y′Y′-N_(a) ′n _(q)′5′   (IIIa)

5′n _(p)-N_(a)-Y Y Y-N_(b)-Z Z Z-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a) ′n _(q)′5′   (IIIb)

5′n _(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(a)′-n _(q)′5′   (IIc)

5′n _(p)-N_(a)-X X X-N_(b)-Y Y Y-N_(b)—ZZZ-N_(a)-n _(q)3′

3′n _(p)′-N_(a)′-X′X′X′-N_(b)′-Y′Y′Y′-N_(b)′-Z′Z′Z′-N_(a)-n _(q)′5′   (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIb), each N_(b) independently represents an oligonucleotide sequence comprising 1-10, 1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIc), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a) independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′ independently represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a), N_(a)′ independently represents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b) and N_(b)′; independently comprises modifications of alternating pattern.

In one embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. In another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications and n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotide a via phosphorothioate linkage. In yet another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p) ′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, and the sense strand is conjugated to one or more C16 (or related) moieties attached through a bivalent or trivalent branched linker (described below). In another embodiment, when the RNAi agent is represented by formula (IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p) ′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties, optionally attached through a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa), the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p) ′>0 and at least one n_(p)′ is linked to a neighboring nucleotide via phosphorothioate linkage, the sense strand comprises at least one phosphorothioate linkage, and the sense strand is conjugated to one or more lipophilic, e.g., C16 (or related) moieties attached through a bivalent or trivalent branched linker.

In one embodiment, the RNAi agent is a multimer containing at least two duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, the RNAi agent is a multimer containing three, four, five, six or more duplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId), wherein the duplexes are connected by a linker. The linker can be cleavable or non-cleavable. Optionally, the multimer further comprises a ligand. Each of the duplexes can target the same gene or two different genes; or each of the duplexes can target same gene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa), (IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, and one or both of the 3′ ends and are optionally conjugated to to a ligand. Each of the agents can target the same gene or two different genes; or each of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used in the methods of the disclosure. Such publications include WO2007/091269, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520; and U.S. Pat. No. 7,858,769, the entire contents of each of which are hereby incorporated herein by reference.

In certain embodiments, the compositions and methods of the disclosure include a vinyl phosphonate (VP) modification of an RNAi agent as described herein. In exemplary embodiments, a vinyl phosphonate of the disclosure has the following structure:

A vinyl phosphonate of the instant disclosure may be attached to either the antisense or the sense strand of a dsRNA of the disclosure. In certain preferred embodiments, a vinyl phosphonate of the instant disclosure is attached to the antisense strand of a dsRNA, optionally at the 5′ end of the antisense strand of the dsRNA.

Vinyl phosphate modifications are also contemplated for the compositions and methods of the instant disclosure. An exemplary vinyl phosphate structure is:

E. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNA interference by incorporating thermally destabilizing modifications in the seed region of the antisense strand (i.e., at positions 2-9 of the 5′-end of the antisense strand) to reduce or inhibit off-target gene silencing. It has been discovered that dsRNAs with an antisense strand comprising at least one thermally destabilizing modification of the duplex within the first 9 nucleotide positions, counting from the 5′ end, of the antisense strand have reduced off-target gene silencing activity. Accordingly, in some embodiments, the antisense strand comprises at least one (e.g., one, two, three, four, five or more) thermally destabilizing modification of the duplex within the first 9 nucleotide positions of the 5′ region of the antisense strand. In some embodiments, one or more thermally destabilizing modification(s) of the duplex is/are located in positions 2-9, or preferably positions 4-8, from the 5′-end of the antisense strand. In some further embodiments, the thermally destabilizing modification(s) of the duplex is/are located at position 6, 7 or 8 from the 5′-end of the antisense strand. In still some further embodiments, the thermally destabilizing modification of the duplex is located at position 7 from the 5′-end of the antisense strand. The term “thermally destabilizing modification(s)” includes modification(s) that would result with a dsRNA with a lower overall melting temperature (Tm) (preferably a Tm with one, two, three or four degrees lower than the Tm of the dsRNA without having such modification(s). In some embodiments, the thermally destabilizing modification of the duplex is located at position 2, 3, 4, 5 or 9 from the 5′-end of the antisense strand.

The thermally destabilizing modifications can include, but are not limited to, abasic modification; mismatch with the opposing nucleotide in the opposing strand; and sugar modification such as 2′-deoxy modification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA) or glycol nucleic acid (GNA).

Exemplified abasic modifications include, but are not limited to the following:

Wherein R═H, Me, Et or OMe; R′═H, Me, Et or OMe; R″═H, Me, Et or OMe

wherein B is a modified or unmodified nucleobase.

Exemplified sugar modifications include, but are not limited to the following:

wherein B is a modified or unmodified nucleobase.

In some embodiments the thermally destabilizing modification of the duplex is selected from the group consisting of:

wherein B is a modified or unmodified nucleobase and the asterisk on each structure represents either R, S or racemic.

The term “acyclic nucleotide” refers to any nucleotide having an acyclic ribose sugar, for example, where any of bonds between the ribose carbons (e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-O4′, or C1′-O4′) is absent or at least one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′, or O4′) are independently or in combination absent from the nucleotide. In some embodiments, acyclic nucleotide is

wherein B is a modified or unmodified nucleobase, R¹ and R² independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refers to unlocked acyclic nucleic acid, wherein any of the bonds of the sugar has been removed, forming an unlocked “sugar” residue. In one example, UNA also encompasses monomers with bonds between C1′-C4′ being removed (i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′ carbons). In another example, the C2′-C3′ bond (i.e. the covalent carbon-carbon bond between the C2′ and C3′ carbons) of the sugar is removed (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059 (1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which are hereby incorporated by reference in their entirety). The acyclic derivative provides greater backbone flexibility without affecting the Watson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or 3′-5′ linkage.

The term ‘GNA’ refers to glycol nucleic acid which is a polymer similar to DNA or RNA but differing in the composition of its “backbone” in that is composed of repeating glycerol units linked by phosphodiester bonds:

The thermally destabilizing modification of the duplex can be mismatches (i.e., noncomplementary base pairs) between the thermally destabilizing nucleotide and the opposing nucleotide in the opposite strand within the dsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T, A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Other mismatch base pairings known in the art are also amenable to the present invention. A mismatch can occur between nucleotides that are either naturally occurring nucleotides or modified nucleotides, i.e., the mismatch base pairing can occur between the nucleobases from respective nucleotides independent of the modifications on the ribose sugars of the nucleotides. In certain embodiments, the dsRNA molecule contains at least one nucleobase in the mismatch pairing that is a 2′-deoxy nucleobase; e.g., the 2′-deoxy nucleobase is in the sense strand.

In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes nucleotides with impaired W-C H-bonding to complementary base on the target mRNA, such as:

More examples of abasic nucleotide, acyclic nucleotide modifications (including UNA and GNA), and mismatch modifications have been described in detail in WO 2011/133876, which is herein incorporated by reference in its entirety.

The thermally destabilizing modifications may also include universal base with reduced or abolished capability to form hydrogen bonds with the opposing bases, and phosphate modifications.

In some embodiments, the thermally destabilizing modification of the duplex includes nucleotides with non-canonical bases such as, but not limited to, nucleobase modifications with impaired or completely abolished capability to form hydrogen bonds with bases in the opposite strand. These nucleobase modifications have been evaluated for destabilization of the central region of the dsRNA duplex as described in WO 2010/0011895, which is herein incorporated by reference in its entirety. Exemplary nucleobase modifications are:

In some embodiments, the thermally destabilizing modification of the duplex in the seed region of the antisense strand includes one or more a-nucleotide complementary to the base on the target mRNA, such as:

wherein R is H, OH, OCH₃, F, NH₂, NHMe, NMe₂ or O-alkyl.

Exemplary phosphate modifications known to decrease the thermal stability of dsRNA duplexes compared to natural phosphodiester linkages are:

R=alkyl

The alkyl for the R group can be a C₁-C₆ alkyl. Specific alkyls for the R group include, but are not limited to methyl, ethyl, propyl, isopropyl, butyl, pentyl and hexyl.

As the skilled artisan will recognize, in view of the functional role of nucleobases is defining specificity of a RNAi agent of the disclosure, while nucleobase modifications can be performed in the various manners as described herein, e.g., to introduce destabilizing modifications into a RNAi agent of the disclosure, e.g., for purpose of enhancing on-target effect relative to off-target effect, the range of modifications available and, in general, present upon RNAi agents of the disclosure tends to be much greater for non-nucleobase modifications, e.g., modifications to sugar groups or phosphate backbones of polyribonucleotides. Such modifications are described in greater detail in other sections of the instant disclosure and are expressly contemplated for RNAi agents of the disclosure, either possessing native nucleobases or modified nucleobases as described above or elsewhere herein.

In addition to the antisense strand comprising a thermally destabilizing modification, the dsRNA can also comprise one or more stabilizing modifications. For example, the dsRNA can comprise at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, the stabilizing modifications all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two stabilizing modifications. The stabilizing modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the stabilizing modification can occur on every nucleotide on the sense strand or antisense strand; each stabilizing modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both stabilizing modification in an alternating pattern. The alternating pattern of the stabilizing modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the stabilizing modifications on the sense strand can have a shift relative to the alternating pattern of the stabilizing modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises stabilizing modifications at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense comprises stabilizing modifications at positions 2, 14, and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one stabilizing modification adjacent to the destabilizing modification. For example, the stabilizing modification can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a stabilizing modification at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.

In some embodiments, the antisense strand comprises at least two stabilizing modifications at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten or more) stabilizing modifications. Without limitations, a stabilizing modification in the sense strand can be present at any positions. In some embodiments, the sense strand comprises stabilizing modifications at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises stabilizing modifications at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises stabilizing modifications at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three, or four stabilizing modifications.

In some embodiments, the sense strand does not comprise a stabilizing modification in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

Exemplary thermally stabilizing modifications include, but are not limited to, 2′-fluoro modifications. Other thermally stabilizing modifications include, but are not limited to, LNA.

In some embodiments, the dsRNA of the disclosure comprises at least four (e.g., four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, the 2′-fluoro nucleotides all can be present in one strand. In some embodiments, both the sense and the antisense strands comprise at least two 2′-fluoro nucleotides. The 2′-fluoro modification can occur on any nucleotide of the sense strand or antisense strand. For instance, the 2′-fluoro modification can occur on every nucleotide on the sense strand or antisense strand; each 2′-fluoro modification can occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both 2′-fluoro modifications in an alternating pattern. The alternating pattern of the 2′-fluoro modifications on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the 2′-fluoro modifications on the sense strand can have a shift relative to the alternating pattern of the 2′-fluoro modifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the antisense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 8, 9, 14, and 16 from the 5′-end. In some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 14, and 16 from the 5′-end. In still some other embodiments, the antisense comprises 2′-fluoro nucleotides at positions 2, 14, and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one 2′-fluoro nucleotide adjacent to the destabilizing modification. For example, the 2′-fluoro nucleotide can be the nucleotide at the 5′-end or the 3′-end of the destabilizing modification, i.e., at position −1 or +1 from the position of the destabilizing modification. In some embodiments, the antisense strand comprises a 2′-fluoro nucleotide at each of the 5′-end and the 3′-end of the destabilizing modification, i.e., positions −1 and +1 from the position of the destabilizing modification.

In some embodiments, the antisense strand comprises at least two 2′-fluoro nucleotides at the 3′-end of the destabilizing modification, i.e., at positions +1 and +2 from the position of the destabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two, three, four, five, six, seven, eight, nine, ten, or more) 2′-fluoro nucleotides. Without limitations, a 2′-fluoro modification in the sense strand can be present at any positions. In some embodiments, the antisense comprises 2′-fluoro nucleotides at positions 7, 10, and 11 from the 5′-end. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions 7, 9, 10, and 11 from the 5′-end. In some embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some other embodiments, the sense strand comprises 2′-fluoro nucleotides at positions opposite or complimentary to positions 11, 12, 13, and 15 of the antisense strand, counting from the 5′-end of the antisense strand. In some embodiments, the sense strand comprises a block of two, three or four 2′-fluoro nucleotides.

In some embodiments, the sense strand does not comprise a 2′-fluoro nucleotide in position opposite or complimentary to the thermally destabilizing modification of the duplex in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises a 21 nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide occurs in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), wherein one end of the dsRNA is blunt, while the other end is comprises a 2 nt overhang, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a blunt end at 5′-end of the antisense strand. Preferably, the 2 nt overhang is at the 3′-end of the antisense.

In some embodiments, the dsRNA molecule of the disclosure comprising a sense and antisense strands, wherein: the sense strand is 25-30 nucleotide residues in length, wherein starting from the 5′ terminal nucleotide (position 1), positions 1 to 23 of said sense strand comprise at least 8 ribonucleotides; antisense strand is 36-66 nucleotide residues in length and, starting from the 3′ terminal nucleotide, at least 8 ribonucleotides in the positions paired with positions 1-23 of sense strand to form a duplex; wherein at least the 3 ‘ terminal nucleotide of antisense strand is unpaired with sense strand, and up to 6 consecutive 3’ terminal nucleotides are unpaired with sense strand, thereby forming a 3′ single stranded overhang of 1-6 nucleotides; wherein the 5′ terminus of antisense strand comprises from 10-30 consecutive nucleotides which are unpaired with sense strand, thereby forming a 10-30 nucleotide single stranded 5′ overhang; wherein at least the sense strand 5′ terminal and 3′ terminal nucleotides are base paired with nucleotides of antisense strand when sense and antisense strands are aligned for maximum complementarity, thereby forming a substantially duplexed region between sense and antisense strands; and antisense strand is sufficiently complementary to a target RNA along at least 19 ribonucleotides of antisense strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell; and wherein the antisense strand contains at least one thermally destabilizing nucleotide, where at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5′-end of the antisense strand). For example, the thermally destabilizing nucleotide occurs between positions opposite or complimentary to positions 14-17 of the 5′-end of the sense strand, and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA comprises a duplex region of 12-30 nucleotide pairs in length.

In some embodiments, the dsRNA molecule of the disclosure comprises a sense and antisense strands, wherein said dsRNA molecule comprises a sense strand having a length which is at least 25 and at most 29 nucleotides and an antisense strand having a length which is at most 30 nucleotides with the sense strand comprises a modified nucleotide that is susceptible to enzymatic degradation at position 11 from the 5′end, wherein the 3′ end of said sense strand and the 5′ end of said antisense strand form a blunt end and said antisense strand is 1˜4 nucleotides longer at its 3′ end than the sense strand, wherein the duplex region which is at least 25 nucleotides in length, and said antisense strand is sufficiently complementary to a target mRNA along at least 19 nt of said antisense strand length to reduce target gene expression when said dsRNA molecule is introduced into a mammalian cell, and wherein dicer cleavage of said dsRNA preferentially results in an siRNA comprising said 3′ end of said antisense strand, thereby reducing expression of the target gene in the mammal, wherein the antisense strand contains at least one thermally destabilizing nucleotide, where the at least one thermally destabilizing nucleotide is in the seed region of the antisense strand (i.e. at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4, or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; and (vi) the dsRNA comprises at least four 2′-fluoro modifications; and (vii) the dsRNA has a duplex region of 12-29 nucleotide pairs in length.

In some embodiments, every nucleotide in the sense strand and antisense strand of the dsRNA molecule may be modified. Each nucleotide may be modified with the same or different modification which can include one or more alteration of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens; alteration of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar; wholesale replacement of the phosphate moiety with “dephospho” linkers; modification or replacement of a naturally occurring base; and replacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modifications occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or a non-linking 0 of a phosphate moiety. In some cases, the modification will occur at all of the subject positions in the nucleic acid but in many cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. e.g., a phosphorothioate modification at a non-linking 0 position may only occur at one or both termini, may only occur in a terminal region, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

It may be possible, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang may be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ position of the ribose sugar with modifications that are known in the art, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or 2′-O-methyl modified instead of the ribosugar of the nucleobase, and modifications in the phosphate group, e.g., phosphorothioate modifications. Overhangs need not be homologous with the target sequence.

In some embodiments, each residue of the sense strand and antisense strand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl, 2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strands can contain more than one modification. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl or 2′-fluoro. It is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.

At least two different modifications are typically present on the sense strand and antisense strand. Those two modifications may be the 2′-deoxy, 2′-O-methyl or 2′-fluoro modifications, acyclic nucleotides or others. In some embodiments, the sense strand and antisense strand each comprises two differently modified nucleotides selected from 2′-O-methyl or 2′-deoxy. In some embodiments, each residue of the sense strand and antisense strand is independently modified with 2′-O-methyl nucleotide, 2′-deoxy nucleotide, 2′-deoxy-2′-fluoro nucleotide, 2′-O-N-methylacetamido (2′-O-NMA) nucleotide, a 2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl (2′-O-AP) nucleotide, or 2′-ara-F nucleotide. Again, it is to be understood that these modifications are in addition to the at least one thermally destabilizing modification of the duplex present in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises modifications of an alternating pattern, particular in the B1, B2, B3, B1′, B2′, B3′, B4′ regions. The term “alternating motif” or “alternative pattern” as used herein refers to a motif having one or more modifications, each modification occurring on alternating nucleotides of one strand. The alternating nucleotide may refer to one per every other nucleotide or one per every three nucleotides, or a similar pattern. For example, if A, B and C each represent one type of modification to the nucleotide, the alternating motif can be “ABABABABABAB . . . ,” “AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,” “AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc. The type of modifications contained in the alternating motif may be the same or different. For example, if A, B, C, D each represent one type of modification on the nucleotide, the alternating pattern, i.e., modifications on every other nucleotide, may be the same, but each of the sense strand or antisense strand can be selected from several possibilities of modifications within the alternating motif such as “ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,” etc.

In some embodiments, the dsRNA molecule of the disclosure comprises the modification pattern for the alternating motif on the sense strand relative to the modification pattern for the alternating motif on the antisense strand is shifted. The shift may be such that the modified group of nucleotides of the sense strand corresponds to a differently modified group of nucleotides of the antisense strand and vice versa. For example, the sense strand when paired with the antisense strand in the dsRNA duplex, the alternating motif in the sense strand may start with “ABABAB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BABABA” from 3′-5′ of the strand within the duplex region. As another example, the alternating motif in the sense strand may start with “AABBAABB” from 5′-3′ of the strand and the alternating motif in the antisense strand may start with “BBAABBAA” from 3′-5′ of the strand within the duplex region, so that there is a complete or partial shift of the modification patterns between the sense strand and the antisense strand.

The dsRNA molecule of the disclosure may further comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. The phosphorothioate or methylphosphonate internucleotide linkage modification may occur on any nucleotide of the sense strand or antisense strand or both in any position of the strand. For instance, the internucleotide linkage modification may occur on every nucleotide on the sense strand or antisense strand; each internucleotide linkage modification may occur in an alternating pattern on the sense strand or antisense strand; or the sense strand or antisense strand comprises both internucleotide linkage modifications in an alternating pattern. The alternating pattern of the internucleotide linkage modification on the sense strand may be the same or different from the antisense strand, and the alternating pattern of the internucleotide linkage modification on the sense strand may have a shift relative to the alternating pattern of the internucleotide linkage modification on the antisense strand.

In some embodiments, the dsRNA molecule comprises the phosphorothioate or methylphosphonate internucleotide linkage modification in the overhang region. For example, the overhang region comprises two nucleotides having a phosphorothioate or methylphosphonate internucleotide linkage between the two nucleotides. Internucleotide linkage modifications also may be made to link the overhang nucleotides with the terminal paired nucleotides within duplex region. For example, at least 2, 3, 4, or all the overhang nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage, and optionally, there may be additional phosphorothioate or methylphosphonate internucleotide linkages linking the overhang nucleotide with a paired nucleotide that is next to the overhang nucleotide. For instance, there may be at least two phosphorothioate internucleotide linkages between the terminal three nucleotides, in which two of the three nucleotides are overhang nucleotides, and the third is a paired nucleotide next to the overhang nucleotide. Preferably, these terminal three nucleotides may be at the 3′-end of the antisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises 1-10 blocks of two to ten phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said sense strand is paired with an antisense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of two phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of three phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of four phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of five phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of six phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of seven phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, or 8 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of eight phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, 4, 5, or 6 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA molecule comprises two blocks of nine phosphorothioate or methylphosphonate internucleotide linkages separated by 1, 2, 3, or 4 phosphate internucleotide linkages, wherein one of the phosphorothioate or methylphosphonate internucleotide linkages is placed at any position in the oligonucleotide sequence and the said antisense strand is paired with a sense strand comprising any combination of phosphorothioate, methylphosphonate and phosphate internucleotide linkages or an antisense strand comprising either phosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s) of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate or methylphosphonate internucleotide linkage at one end or both ends of the sense or antisense strand.

In some embodiments, the dsRNA molecule of the disclosure further comprises one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the internal region of the duplex of each of the sense or antisense strand. For example, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides may be linked through phosphorothioate methylphosphonate internucleotide linkage at position 8-16 of the duplex region counting from the 5′-end of the sense strand; the dsRNA molecule can optionally further comprise one or more phosphorothioate or methylphosphonate internucleotide linkage modification within 1-10 of the termini position(s).

In some embodiments, the dsRNA molecule of the disclosure further comprises one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 1-5 and one to five phosphorothioate or methylphosphonate internucleotide linkage modification(s) within position 18-23 of the sense strand (counting from the 5′-end), and one to five phosphorothioate or methylphosphonate internucleotide linkage modification at positions 1 and 2 and one to five within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate or methylphosphonate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate or methylphosphonate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and two phosphorothioate internucleotide linkage modifications within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modification at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification within position 1-5 (counting from the 5′-end) of the sense strand, and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 (counting from the 5′-end) of the sense strand, and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and one phosphorothioate internucleotide linkage modification within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications within position 1-5 and one phosphorothioate internucleotide linkage modification within position 18-23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications within positions 18-23 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 20 and 21 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 20 and 21 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 21 and 22 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 21 and 22 the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises two phosphorothioate internucleotide linkage modifications at position 1 and 2, and two phosphorothioate internucleotide linkage modifications at position 22 and 23 of the sense strand (counting from the 5′-end), and one phosphorothioate internucleotide linkage modification at positions 1 and one phosphorothioate internucleotide linkage modification at position 21 of the antisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure further comprises one phosphorothioate internucleotide linkage modification at position 1, and one phosphorothioate internucleotide linkage modification at position 21 of the sense strand (counting from the 5′-end), and two phosphorothioate internucleotide linkage modifications at positions 1 and 2 and two phosphorothioate internucleotide linkage modifications at positions 23 and 23 the antisense strand (counting from the 5′-end).

In some embodiments, compound of the disclosure comprises a pattern of backbone chiral centers. In some embodiments, a common pattern of backbone chiral centers comprises at least 5 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 6 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 7 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 8 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 9 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 16 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 17 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 18 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises at least 19 internucleotidic linkages in the Sp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages in the Rp configuration. In some embodiments, a common pattern of backbone chiral centers comprises no more than 8 internucleotidic linkages which are not chiral (as a non-limiting example, a phosphodiester). In some embodiments, a common pattern of backbone chiral centers comprises no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 4 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 3 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 2 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises no more than 1 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 10 internucleotidic linkages in the Sp configuration, and no more than 8 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 11 internucleotidic linkages in the Sp configuration, and no more than 7 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 12 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 13 internucleotidic linkages in the Sp configuration, and no more than 6 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 14 internucleotidic linkages in the Sp configuration, and no more than 5 internucleotidic linkages which are not chiral. In some embodiments, a common pattern of backbone chiral centers comprises at least 15 internucleotidic linkages in the Sp configuration, and no more than 4 internucleotidic linkages which are not chiral. In some embodiments, the internucleotidic linkages in the Sp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages in the Rp configuration are optionally contiguous or not contiguous. In some embodiments, the internucleotidic linkages which are not chiral are optionally contiguous or not contiguous.

In some embodiments, compound of the disclosure comprises a block is a stereochemistry block. In some embodiments, a block is an Rp block in that each internucleotidic linkage of the block is Rp. In some embodiments, a 5′-block is an Rp block. In some embodiments, a 3′-block is an Rp block. In some embodiments, a block is an Sp block in that each internucleotidic linkage of the block is Sp. In some embodiments, a 5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block. In some embodiments, provided oligonucleotides comprise both Rp and Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Rp but no Sp blocks. In some embodiments, provided oligonucleotides comprise one or more Sp but no Rp blocks. In some embodiments, provided oligonucleotides comprise one or more PO blocks wherein each internucleotidic linkage in a natural phosphate linkage.

In some embodiments, compound of the disclosure comprises a 5′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 5′-block comprises 4 or more nucleoside units. In some embodiments, a 5′-block comprises 5 or more nucleoside units. In some embodiments, a 5′-block comprises 6 or more nucleoside units. In some embodiments, a 5′-block comprises 7 or more nucleoside units. In some embodiments, a 3′-block is an Sp block wherein each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a modified internucleotidic linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block is an Sp block wherein each of internucleotidic linkage is a phosphorothioate linkage and each sugar moiety comprises a 2′-F modification. In some embodiments, a 3′-block comprises 4 or more nucleoside units. In some embodiments, a 3′-block comprises 5 or more nucleoside units. In some embodiments, a 3′-block comprises 6 or more nucleoside units. In some embodiments, a 3′-block comprises 7 or more nucleoside units.

In some embodiments, compound of the disclosure comprises a type of nucleoside in a region or an oligonucleotide is followed by a specific type of internucleotidic linkage, e.g., natural phosphate linkage, modified internucleotidic linkage, Rp chiral internucleotidic linkage, Sp chiral internucleotidic linkage, etc. In some embodiments, A is followed by Sp. In some embodiments, A is followed by Rp. In some embodiments, A is followed by natural phosphate linkage (PO). In some embodiments, U is followed by Sp. In some embodiments, U is followed by Rp. In some embodiments, U is followed by natural phosphate linkage (PO). In some embodiments, C is followed by Sp. In some embodiments, C is followed by Rp. In some embodiments, C is followed by natural phosphate linkage (PO). In some embodiments, G is followed by Sp. In some embodiments, G is followed by Rp. In some embodiments, G is followed by natural phosphate linkage (PO). In some embodiments, C and U are followed by Sp. In some embodiments, C and U are followed by Rp. In some embodiments, C and U are followed by natural phosphate linkage (PO). In some embodiments, A and G are followed by Sp. In some embodiments, A and G are followed by Rp.

In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.

In some embodiments, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (iv) the sense strand comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2′-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.

In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six, seven or all eight) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5, or 6 2′-fluoro modifications; (ii) the antisense comprises 1, 2, 3, 4, or 5 phosphorothioate internucleotide linkages; (iii) the sense strand is conjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (vi) the dsRNA comprises at least four 2′-fluoro modifications; (vii) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNA has a blunt end at 5′-end of the antisense strand.

In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, and between nucleotide positions 2 and 3, the antisense strand comprises phosphorothioate internucleotide linkages between nucleotide positions 1 and 2, between nucleotide positions 2 and 3, between nucleotide positions 21 and 22, and between nucleotide positions 22 and 23, wherein the antisense strand contains at least one thermally destabilizing modification of the duplex located in the seed region of the antisense strand (i.e., at position 2-9 of the 5′-end of the antisense strand), and wherein the dsRNA optionally further has at least one (e.g., one, two, three, four, five, six or all seven) of the following characteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoro modifications; (ii) the sense strand is conjugated with a ligand; (iii) the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (iv) the sense strand comprises 3, 4 or 5 phosphorothioate internucleotide linkages; (v) the dsRNA comprises at least four 2′-fluoro modifications; (vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs in length; and (vii) the dsRNA has a blunt end at 5′-end of the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises mismatch(es) with the target, within the duplex, or combinations thereof. The mismatch can occur in the overhang region or the duplex region. The base pair can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation: A:U is preferred over G:C; G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine). Mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings; and pairings which include a universal base are preferred over canonical pairings.

In some embodiments, the dsRNA molecule of the disclosure comprises at least one of the first 1, 2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end of the antisense strand can be chosen independently from the group of: A:U, G:U, I:C, and mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base, to promote the dissociation of the antisense strand at the 5′-end of the duplex.

In some embodiments, the nucleotide at the 1 position within the duplex region from the 5′-end in the antisense strand is selected from the group consisting of A, dA, dU, U, and dT. Alternatively, at least one of the first 1, 2 or 3 base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair. For example, the first base pair within the duplex region from the 5′-end of the antisense strand is an AU base pair.

It was found that introducing 4′-modified or 5′-modified nucleotide to the 3′-end of a phosphodiester (PO), phosphorothioate (PS), or phosphorodithioate (PS2) linkage of a dinucleotide at any position of single stranded or double stranded oligonucleotide can exert steric effect to the internucleotide linkage and, hence, protecting or stabilizing it against nucleases.

In some embodiments, 5′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 5′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 5′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-modified nucleoside is introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. For instance, a 4′-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The alkyl group at the 4′ position of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer. Alternatively, a 4′-O-alkylated nucleoside may be introduced at the 3′-end of a dinucleotide at any position of single stranded or double stranded siRNA. The 4′-O-alkyl of the ribose sugar can be racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 5′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 4′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 4′-O-alkylated nucleoside is introduced at any position on the sense strand or antisense strand of a dsRNA, and such modification maintains or improves potency of the dsRNA. The 5′-alkyl can be either racemic or chirally pure R or S isomer. An exemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, the dsRNA molecule of the disclosure can comprise 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S). For example, the 2′-5′ linkages modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

In another embodiment, the dsRNA molecule of the disclosure can comprise L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe). For example, these L sugars modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

Various publications describe multimeric siRNA which can all be used with the dsRNA of the disclosure. Such publications include WO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887, and WO2011/031520 which are hereby incorporated by their entirely.

As described in more detail below, the RNAi agent that contains conjugations of one or more carbohydrate moieties to an RNAi agent can optimize one or more properties of the RNAi agent. In many cases, the carbohydrate moiety will be attached to a modified subunit of the RNAi agent. For example, the ribose sugar of one or more ribonucleotide subunits of a dsRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier to which is attached a carbohydrate ligand. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. The carriers include (i) at least one “backbone attachment point,” preferably two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” (TAP) in some embodiments refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a carbohydrate, e.g. monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide and polysaccharide. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, the cyclic carrier will often include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein the carrier can be cyclic group or acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methods of the disclosure is an agent selected from the group of agents listed in any one of Tables 2-7, 9, and 10. These agents may further comprise a ligand, such as one or more lipophilic moieties, one or more GalNAc derivatives, or both of one of more lipophilic moieties and one or more GalNAc derivatives.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the iRNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA, e.g., into a cell. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

In certain embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In some embodiments, a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Typical ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an a helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic. In certain embodiments, the ligand is a multivalent galactose, e.g., an N-acetyl-galactosamine.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated iRNAs of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems® (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In certain embodiments, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule can typically bind a serum protein, such as human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, naproxen or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid-based ligand can be used to modulate, e.g., control (e.g., inhibit) the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In certain embodiments, the lipid-based ligand binds HSA. For example, the ligand can bind HSA with a sufficient affinity such that distribution of the conjugate to a non-kidney tissue is enhanced. However, the affinity is typically not so strong that the HSA-ligand binding cannot be reversed.

In certain embodiments, the lipid-based ligand binds HSA weakly or not at all, such that distribution of the conjugate to the kidney is enhanced. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid-based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, such as a helical cell-permeation agent. In certain embodiments, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is typically an α-helical agent and can have a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 11). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 12)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 13)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 14)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OB OC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Typically, the peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glycosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

An RGD peptide moiety can be used to target a particular cell type, e.g., a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an dsRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). Typically, the RGD peptide will facilitate targeting of an iRNA agent to the kidney. The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an a-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and tri-saccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In certain embodiments, a carbohydrate conjugate comprises a monosaccharide.

In certain embodiments, the monosaccharide is an N-acetylgalactosamine (GalNAc). GalNAc conjugates, which comprise one or more N-acetylgalactosamine (GalNAc) derivatives, are described, for example, in U.S. Pat. No. 8,106,022, the entire content of which is hereby incorporated herein by reference. In some embodiments, the GalNAc conjugate serves as a ligand that targets the iRNA to particular cells. In some embodiments, the GalNAc conjugate targets the iRNA to liver cells, e.g., by serving as a ligand for the asialoglycoprotein receptor of liver cells (e.g., hepatocytes).

In some embodiments, the carbohydrate conjugate comprises one or more GalNAc derivatives. The GalNAc derivatives may be attached via a linker, e.g., a bivalent or trivalent branched linker. In some embodiments the GalNAc conjugate is conjugated to the 3′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 3′ end of the sense strand) via a linker, e.g., a linker as described herein. In some embodiments the GalNAc conjugate is conjugated to the 5′ end of the sense strand. In some embodiments, the GalNAc conjugate is conjugated to the iRNA agent (e.g., to the 5′ end of the sense strand) via a linker, e.g., a linker as described herein.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker. The hairpin loop may also be formed by an extended overhang in one strand of the duplex.

In some embodiments, the GalNAc conjugate is

In some embodiments, the RNAi agent is attached to the carbohydrate conjugate via a linker as shown in the following schematic, wherein X is O or S

In some embodiments, the RNAi agent is conjugated to L96 as defined in Table 1 and shown below:

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

In certain embodiments, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In certain embodiments, the monosaccharide is an N-acetylgalactosamine, such as

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

-   -   when one of X or Y is an oligonucleotide, the other is a         hydrogen.

In some embodiments, a suitable ligand is a ligand disclosed in WO 2019/055633, the entire contents of which are incorporated herein by reference. In one embodiment the ligand comprises the structure below:

In certain embodiments, the RNAi agents of the disclosure may include GalNAc ligands, even if such GalNAc ligands are currently projected to be of limited value for the preferred pulmonary system delivery route(s) of the instant disclosure.

In certain embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a monovalent linker. In some embodiments, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a bivalent linker. In yet other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a trivalent linker. In other embodiments of the invention, the GalNAc or GalNAc derivative is attached to an iRNA agent of the invention via a tetravalent linker.

In certain embodiments, the double stranded RNAi agents of the invention comprise one GalNAc or GalNAc derivative attached to the iRNA agent, e.g., the 5′ end of the sense strand of a dsRNA agent, or the 5′ end of one or both sense strands of a dual targeting RNAi agent as described herein. In certain embodiments, the double stranded RNAi agents of the invention comprise a plurality (e.g., 2, 3, 4, 5, or 6) GalNAc or GalNAc derivatives, each independently attached to a plurality of nucleotides of the double stranded RNAi agent through a plurality of monovalent linkers.

In some embodiments, for example, when the two strands of an iRNA agent of the invention are part of one larger molecule connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming a hairpin loop comprising, a plurality of unpaired nucleotides, each unpaired nucleotide within the hairpin loop may independently comprise a GalNAc or GalNAc derivative attached via a monovalent linker.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator or a cell permeation peptide.

Additional carbohydrate conjugates and linkers suitable for use in the present invention include those described in WO 2014/179620 and WO 2014/179627, the entire contents of each of which are incorporated herein by reference.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non-cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In certain embodiments, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-16, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In certain embodiments, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In certain embodiments, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In certain embodiments, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Cleavable Linking Groups

In certain embodiments, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleavable Linking Groups

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In some embodiments, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more “GalNAc” (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In certain embodiments, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XLV)-(XLVI):

-   -   wherein:     -   q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent         independently for each occurrence 0-20 and wherein the repeating         unit can be the same or different;     -   P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B),         P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A),         T^(5B), T^(5C) are each independently for each occurrence         absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O;     -   Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B),         Q^(5C) are independently for each occurrence absent, alkylene,         substituted alkylene wherein one or more methylenes can be         interrupted or terminated by one or more of O, S, S(O), SO₂, N(R         N), C(R′)═C(R″), C≡C or C(O);     -   R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B),         R^(5C) are each independently for each occurrence absent, NH, O,         S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO,         CH═N-O,

or heterocyclyl;

-   -   L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B)         and L^(5C) represent the ligand; i.e. each independently for         each occurrence a monosaccharide (such as GalNAc), disaccharide,         trisaccharide, tetrasaccharide, oligosaccharide, or         polysaccharide; and R^(a) is H or amino acid side chain.         Trivalent conjugating GalNAc derivatives are particularly useful         for use with RNAi agents for inhibiting the expression of a         target gene, such as those of formula (XLIX):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,         such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II, VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; and 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNA agents, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

V. Delivery of an RNAi Agent of the Disclosure

The delivery of a RNAi agent of the disclosure to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a MUC5B-associated disorder, e.g., a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF), e.g., a subject having or at risk of developing or at risk of having a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF), can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an RNAi agent of the disclosure either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an RNAi agent, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the RNAi agent. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with a RNAi agent of the disclosure (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an RNAi agent include, for example, biological stability of the delivered agent, prevention of non-specific effects, and accumulation of the delivered agent in the target tissue. The non-specific effects of an RNAi agent can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the RNAi agent to be administered. Several studies have shown successful knockdown of gene products when an RNAi agent is administered locally. For example, pulmonary delivery, e.g., inhalation, of a dsRNA, e.g., SOD1, has been shown to effectively knockdown gene and protein expression in lung tissue and that there is excellent uptake of the dsRNA by the bronchioles and alveoli of the lung. Intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M I et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were also both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering a RNAi agent systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the RNAi agent to the target tissue and avoid undesirable off-target effects (e.g., without wishing to be bound by theory, use of GNAs as described herein has been identified to destabilize the seed region of a dsRNA, resulting in enhanced preference of such dsRNAs for on-target effectiveness, relative to off-target effects, as such off-target effects are significantly weakened by such seed region destabilization). RNAi agents can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, a RNAi agent directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an RNAi agent to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the RNAi agent can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of molecule RNAi agent (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an RNAi agent by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an RNAi agent, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an RNAi agent. The formation of vesicles or micelles further prevents degradation of the RNAi agent when administered systemically. Methods for making and administering cationic-RNAi agent complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al. (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of RNAi agents include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, a RNAi agent forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of RNAi agents and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

Certain aspects of the instant disclosure relate to a method of reducing the expression of a MUC5B gene in a cell, comprising contacting said cell with the double-stranded RNAi agent of the disclosure. In one embodiment, the cell is a hepatic cell, optionally a hepatocyte. In one embodiment, the cell is an extrahepatic cell, optionally a pulmonary cell.

In one embodiment, the cell is present in an organ or tissues of the respiratory system, including, but not limited to, bronchus, bronchiole, alveoli, epithelium including nasal and respiratory epithelium, ciliated epithelium, and goblet cells; pneumocytes, both type I and type II, macrophages, peritubular interstitium, macrophages, adipose tissue, e.g., mediastinal adipose tissue, pulmonary cell. neuronal cells, e.g., in the pulmonary neuroal plexus, club cells, clara cells, neutrophils, both resident and transient, and oral mucosa.

In certain embodiments, the RNAi agent is taken up on one or more tissue or cell types present in organs outside of the respiratory system, e.g., liver, kidney.

Another aspect of the disclosure relates to a method of reducing the expression and/or activity of a MUC5B gene in a subject, comprising administering to the subject the double-stranded RNAi agent of the disclosure.

Another aspect of the disclosure relates to a method of treating a subject having a MUC5B-associated disorder or at risk of having or at risk of developing a MUC5B-associated disorder, comprising administering to the subject a therapeutically effective amount of the double-stranded RNAi agent of the disclosure, thereby treating the subject. In some embodiments, the MUC5B-associated disorder comprises a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF).

In one embodiment, the double-stranded RNAi agent is administered subcutaneously.

In one embodiment, the double-stranded RNAi agent is administered by pulmonary system administration, e.g., intranasal administration, or oral inhalative administration.

In one embodiment, the double-stranded RNAi agent is administered intranasally.

By pulmonary system administration, e.g., intranasal administration or oral inhalative administration, of the double-stranded RNAi agent, the method can reduce the expression of an MUC5B target gene in a pulmonary system tissue, e.g., a nasopharynx tissue, an oropharynx tissue, a laryngopharynx tissue, a larynx tissue, a trachea tissue, a carina tissue, a bronchi tissue, a bronchiole tissue, or an alveoli tissue.

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to modified siRNA compounds. It may be understood, however, that these formulations, compositions and methods can be practiced with other siRNA compounds, e.g., unmodified siRNA compounds, and such practice is within the disclosure. A composition that includes a RNAi agent can be delivered to a subject by a variety of routes. Exemplary routes include pulmonary system, intravenous, intraventricular, topical, rectal, anal, vaginal, nasal, and ocular.

The RNAi agents of the disclosure can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of RNAi agent and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral, or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal, or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the RNAi agent in powder or aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the RNAi agent and mechanically introducing the RNA.

Compositions for pulmonary system delivery may include aqueous solutions, e.g., for intranasal or oral inhalative administration, suitable carriers composed of, e.g., lipids (liposomes, niosomes, microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration. Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorbtion enhancers and other suitable additives.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves, and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening or flavoring agents can be added.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents, and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes may be controlled to render the preparation isotonic.

In one embodiment, the administration of the siRNA compound, e.g., a double-stranded siRNA compound, or ssiRNA compound, composition is parenteral, e.g., intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary system, intranasal, urethral, or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

Pulmonary System Administration

In one embodiment, the double-stranded RNAi agent is administered by pulmonary system administration. The pulmonary system includes the upper pulmonary system and the lower pulmonary system. The upper pulmonary system includes the nose and the pharynx. The pharynx includes the nasopharynx, oropharynx, and laryngopharynx. The lower pulmonary system includes the larynx, trachea, carina, bronchi, bronchioles, and alveoli.

Pulmonary system administration may be intranasal administration or oral inhalative administration. Such administration permits both systemic and local delivery of the double stranded RNAi agents of the invention.

Intranasal administration may include instilling or insufflating a double stranded RNAi agent into the nasal cavity with syringes or droppers by applying a few drops at a time or via atomization. Suitable dosage forms for intranasal administration include drops, powders, nebulized mists, and sprays. Nasal delivery devices include, but not limited to, vapor inhaler, nasal dropper, spray bottle, metered dose spray pump, gas driven spray atomizer, nebulizer, mechanical powder sprayer, breath actuated inhaler, and insufflator. Devices for delivery deeper into the respiratory system, e.g., into the lung, include nebulizer, pressured metered-dose inhaler, dry powder inhaler, and thermal vaporization aerosol device. Devices for delivery by inhalation are available from commercial suppliers. Devices can be fixed or variable dose, single or multidose, disposable or reusable depending on, for example, the disease or disorder to be prevented or treated, the volume of the agent to be delivered, the frequency of delivery of the agent, and other considerations in the art.

Oral inhalative administration may include use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI), to deliver a double stranded RNAi agent to the pulmonary system. Suitable dosage forms for oral inhalative administration include powders and solutions. Suitable devices for oral inhalative administration include nebulizers, metered-dose inhalers, and dry powder inhalers. Dry powder inhalers are of the most popular devices used to deliver drugs, especially proteins to the lungs. Exemplary commercially available dry powder inhalers include Spinhaler (Fisons Pharmaceuticals, Rochester, NY) and Rotahaler (GSK, RTP, NC). Several types of nebulizers are available, namely jet nebulizers, ultrasonic nebulizers, vibrating mesh nebulizers. Jet nebulizers are driven by compressed air. Ultrasonic nebulizers use a piezoelectric transducer in order to create droplets from an open liquid reservoir. Vibrating mesh nebulizers use perforated membranes actuated by an annular piezoelement to vibrate in resonant bending mode. The holes in the membrane have a large cross-section size on the liquid supply side and a narrow cross-section size on the side from where the droplets emerge. Depending on the therapeutic application, the hole sizes and number of holes can be adjusted. Selection of a suitable device depends on parameters, such as nature of the drug and its formulation, the site of action, and pathophysiology of the lung. Aqueous suspensions and solutions are nebulized effectively. Aerosols based on mechanically generated vibration mesh technologies also have been used successfully to deliver proteins to lungs.

The amount of RNAi agent for pulmonary system administration may vary from one target gene to another target gene and the appropriate amount that has to be applied may have to be determined individually for each target gene. Typically, this amount ranges from 10 μg to 2 mg, preferably 50 μg to 1500 μg, more preferably 100 μg to 1000 μg.

Vector Encoded RNAi Agents of the Disclosure

RNAi agents targeting the MUC5B gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; WO 00/22113, WO 00/22114, and U.S. Pat. No. 6,054,299). Expression is preferably sustained (months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of a RNAi agent can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively, each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

RNAi agent expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of a RNAi agent as described herein. Delivery of RNAi agent expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of a RNAi agent will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the RNAi agent in target cells. Other aspects to consider for vectors and constructs are known in the art.

VI. Pharmaceutical Compositions of the Invention

The present disclosure also includes pharmaceutical compositions and formulations which include the RNAi agents of the disclosure. In one embodiment, provided herein are pharmaceutical compositions containing an RNAi agent, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the RNAi agent are useful for treating a subject who would benefit from inhibiting or reducing the expression of a MUC5B gene, e.g., a subject having a MUC5B-associated disorder, e.g., a subject having or at risk of having or at risk of developing a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF). Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for direct delivery into the pulmonary system by intranasal administration or oral inhalative administration, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal delivery. Another example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV), intramuscular (IM), or for subcutaneous (subQ) delivery.

In some embodiments, the pharmaceutical compositions of the invention are pyrogen free or non-pyrogenic.

The pharmaceutical compositions of the disclosure may be administered in dosages sufficient to inhibit expression of a MUC5B gene. In general, a suitable dose of an RNAi agent of the disclosure will be a flat dose in the range of about 0.001 to about 200.0 mg about once per month to about once per year, typically about once per quarter (i.e., about once every three months) to about once per year, generally a flat dose in the range of about 1 to 50 mg about once per month to about once per year, typically about once per quarter to about once per year. In certain embodiments, the dose will be a fixed dose, e.g., a fixed dose of about 25 ug to about 5 mg.

A repeat-dose regimen may include administration of a therapeutic amount of a RNAi agent on a regular basis, such as monthly to once every six months. In certain embodiments, the RNAi agent is administered about once per quarter (i.e., about once every three months) to about twice per year, particularly for treatment of a chronic disease.

After an initial treatment regimen (e.g., loading dose), of once per day, twice per week, once per week, the treatments can be administered on a less frequent basis.

In other embodiments, a single dose of the pharmaceutical compositions can be long lasting, such that subsequent doses are administered at not more than 1, 2, 3, or 4 or more month intervals. In some embodiments of the disclosure, a single dose of the pharmaceutical compositions of the disclosure is administered once per month. In other embodiments of the disclosure, a single dose of the pharmaceutical compositions of the disclosure is administered once per quarter to twice per year.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments.

Advances in mouse genetics have generated a number of mouse models for the study of various MUC5B-associated diseases that would benefit from reduction in the expression of MUC5B. Such models can be used for in vivo testing of RNAi agents, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, the mouse models described elsewhere herein.

The pharmaceutical compositions of the present disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary system administration by intranasal administration or oral inhalative administration, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The RNAi agents can be delivered in a manner to target a particular tissue, such as the liver, the lung (e.g., bronchioles, alveoli, or bronchus of the lung), or both the liver and lung.

Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the RNAi agents featured in the disclosure are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). RNAi agents featured in the disclosure can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, RNAi agents can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. RNAi Agent Formulations Comprising Membranous Molecular Assemblies

A RNAi agent for use in the compositions and methods of the disclosure can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the RNAi agent composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the RNAi agent composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the RNAi agent are delivered into the cell where the RNAi agent can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the RNAi agent to particular cell types.

A liposome containing an RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid or phosphatidylcholine or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Indiana) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wisconsin) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, California) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Maryland). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.

Liposomes that include RNAi agents can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present disclosure are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application number PCT/US2007/080331, filed Oct. 3, 2007, also describes formulations that are amenable to the present disclosure.

Transfersomes, yet another type of liposomes, are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as those described herein, particularlay in emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The RNAi agent for use in the methods of the disclosure can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

Lipid Particles

RNAi agents, e.g., dsRNAs of in the disclosure may be fully encapsulated in a lipid formulation, e.g., a LNP, or other nucleic acid-lipid particle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipid particle. LNPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). LNPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). LNPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in WO 00/03683. The particles of the present disclosure typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present disclosure are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; United States Patent publication No. 2010/0324120 and WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the disclosure.

Certain specific LNP formulations for delivery of RNAi agents have been described in the art, including, e.g., “LNP01” formulations as described in, e.g., WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are identified in the table below.

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-1 1,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-CDMA dimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA ~ 7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG-CDMA dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~ 7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~ 6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~ 11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~ 6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~ 11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMG di((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH- Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG-DMG tetraen-19-yl 4-(dimethylamino)butanoate 50/10/38.5/1.5 (MC3) Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2- 50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin-1- Lipid:siRNA 10:1 yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-CDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in WO 2009/127060, which is hereby incorporated by reference. XTC comprising formulations are described in WO 2010/088537, the entire contents of which are hereby incorporated herein by reference. MC3 comprising formulations are described, e.g., in United States Patent Publication No. 2010/0324120, the entire contents of which are hereby incorporated by reference. ALNY-100 comprising formulations are described in WO 2010/054406, the entire contents of which are hereby incorporated herein by reference. C12-200 comprising formulations are described in WO 2010/129709, the entire contents of which are hereby incorporated herein by reference.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the disclosure are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids or esters or salts thereof, bile acids or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the disclosure can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, U.S. 2003/0027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions for pulmonary system delivery may include aqueous solutions, e.g., for intranasal or oral inhalative administration, suitable carriers composed of, e.g., lipids (liposomes, niosomes, microemulsions, lipidic micelles, solid lipid nanoparticles) or polymers (polymer micelles, dendrimers, polymeric nanoparticles, nonogels, nanocapsules), adjuvant, e.g., for oral inhalative administration. Aqueous compositions may be sterile and may optionally contain buffers, diluents, absorbtion enhancers and other suitable additives.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the brain when treating APP-associated diseases or disorders.

The pharmaceutical formulations of the present disclosure, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present disclosure can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present disclosure can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.

Additional Formulations

i. Emulsions

The compositions of the present disclosure can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise, a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present disclosure, the compositions of RNAi agents and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically, microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used, and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, NY; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or RNAi agents. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present disclosure will facilitate the increased systemic absorption of RNAi agents and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of RNAi agents and nucleic acids.

Microemulsions of the present disclosure can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the RNAi agents and nucleic acids of the present disclosure. Penetration enhancers used in the microemulsions of the present disclosure can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

An RNAi agent of the disclosure may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present disclosure employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly RNAi agents, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of RNAi agents through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, M A, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, NY, 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present disclosure, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of RNAi agents through the mucosa is enhanced. With regards to their use as penetration enhancers in the present disclosure, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, M A, 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rd., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of RNAi agents through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of RNAi agents at the cellular level can also be added to the pharmaceutical and other compositions of the present disclosure. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (WO 97/30731), are also known to enhance the cellular uptake of dsRNAs.

Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present disclosure. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

vii. Other Components

The compositions of the present disclosure can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the disclosure include (a) one or more RNAi agents and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a MUC5B-associated disorder. Examples of such agents include, but are not limited to an antiviral agent, an immune stimulator, a therapeutic vaccine, a viral entry inhibitor, and a combination of any of the foregoing.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the disclosure lies generally within a range of circulating concentrations that include the ED 50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the disclosure, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC 50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the RNAi agents featured in the disclosure can be administered in combination with other known agents effective in treatment of pathological processes mediated by nucleotide repeat expression. In any event, the administering physician can adjust the amount and timing of RNAi agent administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VII. Kits

In certain aspects, the instant disclosure provides kits that include a suitable container containing a pharmaceutical formulation of a siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, (e.g., a precursor, e.g., a larger siRNA compound which can be processed into a siRNA compound, or a DNA which encodes an siRNA compound, e.g., a double-stranded siRNA compound, or siRNA compound, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for a siRNA compound preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device, such as a device suitable for pulmonary administration, e.g., a device suitable for oral inhalative administration including nebulizers, metered-dose inhalers, and dry powder inhalers.

VIII. Methods for Inhibiting MUC5B Expression

The present disclosure also provides methods of inhibiting expression of a MUC5B gene in a cell. The methods include contacting a cell with an RNAi agent, e.g., double stranded RNAi agent, in an amount effective to inhibit expression of a MUC5B gene in the cell, thereby inhibiting expression of MUC5B in the cell. In certain embodiments of the disclosure, expression of a MUC5B gene is inhibited preferentially in the pulmonary system (e.g., lung, bronchial, alveoli) cells. In other embodiments of the disclosure, expression of a MUC5B gene is inhibited preferentially in the liver (e.g., hepatocytes). In certain embodiments of the disclosure, expression of a MUC5B gene is inhibited in the pulmonary system (e.g., lung, bronchial, alveoli) cells and in liver (e.g., hepatocytes) cells.

Contacting of a cell with a RNAi agent, e.g., a double stranded RNAi agent, may be done in vitro or in vivo. Contacting a cell in vivo with the RNAi agent includes contacting a cell or group of cells within a subject, e.g., a human subject, with the RNAi agent. Combinations of in vitro and in vivo methods of contacting a cell are also possible.

Contacting a cell may be direct or indirect, as discussed above. Furthermore, contacting a cell may be accomplished via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a lipophilic moiety, e.g., a C16, and/or a carbohydrate moiety, e.g., a GalNAc ligand, or any other ligand that directs the RNAi agent to a site of interest. In certain embodiments, the ligand is not a cholesterol moiety. In certain embodiments, the RNAi agent does not include a targeting ligand.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition. In certain embodiments, a level of inhibition, e.g., for an RNAi agent of the instant disclosure, can be assessed in cell culture conditions, e.g., wherein cells in cell culture are transfected via Lipofectamine™-mediated transfection at a concentration in the vicinity of a cell of 10 nM or less, 1 nM or less, etc. Knockdown of a given RNAi agent can be determined via comparison of pre-treated levels in cell culture versus post-treated levels in cell culture, optionally also comparing against cells treated in parallel with a scrambled or other form of control RNAi agent. Knockdown in cell culture of, e.g., preferably 50% or more, can thereby be identified as indicative of “inhibiting” or “reducing”, “downregulating” or “suppressing”, etc. having occurred. It is expressly contemplated that assessment of targeted mRNA or encoded protein levels (and therefore an extent of “inhibiting”, etc. caused by a RNAi agent of the disclosure) can also be assessed in in vivo systems for the RNAi agents of the instant disclosure, under properly controlled conditions as described in the art.

The phrase “inhibiting expression of a MUC5B gene” or “inhibiting expression of MUC5B,” as used herein, includes inhibition of expression of any MUC5B gene (such as, e.g., a mouse MUC5B gene, a rat MUC5B gene, a monkey MUC5B gene, or a human MUC5B gene) as well as variants or mutants of a MUC5B gene that encode a MUC5B protein. Thus, the MUC5B gene may be a wild-type MUC5B gene, a mutant MUC5B gene, or a transgenic MUC5B gene in the context of a genetically manipulated cell, group of cells, or organism.

“Inhibiting expression of a MUC5B gene” includes any level of inhibition of a MUC5B gene, e.g., at least partial suppression of the expression of a MUC5B gene, such as an inhibition by at least 20%. In certain embodiments, inhibition is by at least 30%, at least 40%, preferably at least 50%, at least about 60%, at least 70%, at least about 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%; or to below the level of detection of the assay method. In a preferred method, inhibition is measured at a 10 nM concentration of the siRNA using the luciferase assay provided in Example 1.

The expression of a MUC5B gene may be assessed based on the level of any variable associated with MUC5B gene expression, e.g., MUC5B mRNA level or MUC5B protein level.

Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In some embodiments of the methods of the disclosure, expression of a MUC5B gene is inhibited by at least 20%, 30%, 40%, preferably at least 50%, 60%, 70%, 80%, 85%, 90%, or 95%, or to below the level of detection of the assay. In certain embodiments, the methods include a clinically relevant inhibition of expression of MUC5B, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of a MUC5B gene.

Inhibition of the expression of a MUC5B gene may be manifested by a reduction of the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample derived from a subject) in which a MUC5B gene is transcribed and which has or have been treated (e.g., by contacting the cell or cells with a RNAi agent of the disclosure, or by administering a RNAi agent of the disclosure to a subject in which the cells are or were present) such that the expression of a MUC5B gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has not or have not been so treated (control cell(s) not treated with a RNAi agent or not treated with a RNAi agent targeted to the genome of interest). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}{in}{control}{cells}} \right) - \left( {{mRNA}{in}{treated}{cells}} \right)}{\left( {{mRNA}{in}{control}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of a MUC5B gene may be assessed in terms of a reduction of a parameter that is functionally linked to a MUC5B gene expression, e.g., MUC5B protein expression, S protein priming, efficiency of viral entry, viral load. MUC5B gene silencing may be determined in any cell expressing a MUC5B gene, either endogenous or heterologous from an expression construct, and by any assay known in the art.

Inhibition of the expression of a MUC5B protein may be manifested by a reduction in the level of the MUC5B protein that is expressed by a cell or group of cells (e.g., the level of protein expressed in a sample derived from a subject). As explained above, for the assessment of genome suppression, the inhibition of protein expression levels in a treated cell or group of cells may similarly be expressed as a percentage of the level of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess the inhibition of the expression of a MUC5B gene includes a cell or group of cells that has not yet been contacted with an RNAi agent of the disclosure. For example, the control cell or group of cells may be derived from an individual subject (e.g., a human or animal subject) prior to treatment of the subject with an RNAi agent.

The level of MUC5B mRNA that is expressed by a cell or group of cells may be determined using any method known in the art for assessing RNA expression. In one embodiment, the level of expression of MUC5B in a sample is determined by detecting a transcribed polynucleotide, or portion thereof, e.g., mRNA of the MUC5B gene. RNA may be extracted from cells using RNA extraction techniques including, for example, using acid phenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNeasy™ RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix, Switzerland). Typical assay formats utilizing ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, northern blotting, in situ hybridization, and microarray analysis. Circulating MUC5B mRNA may be detected using methods the described in WO2012/177906, the entire contents of which are hereby incorporated herein by reference.

In some embodiments, the level of expression of MUC5B is determined using a nucleic acid probe. The term “probe”, as used herein, refers to any molecule that is capable of selectively binding to a specific MUC5B nucleic acid or protein, or fragment thereof. Probes can be synthesized by one of skill in the art, or derived from appropriate biological preparations. Probes may be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

Isolated mRNA can be used in hybridization or amplification assays that include, but are not limited to, Southern or northern analyses, polymerase chain reaction (PCR) analyses and probe arrays. One method for the determination of RNA levels involves contacting the isolated RNA with a nucleic acid molecule (probe) that can hybridize to MUC5B RNA. In one embodiment, the RNA is immobilized on a solid surface and contacted with a probe, for example by running the isolated RNA on an agarose gel and transferring the RNA from the gel to a membrane, such as nitrocellulose. In an alternative embodiment, the probe(s) are immobilized on a solid surface and the RNA is contacted with the probe(s), for example, in an Affymetrix gene chip array. A skilled artisan can readily adapt known RNA detection methods for use in determining the level of MUC5B mRNA.

An alternative method for determining the level of expression of MUC5B in a sample involves the process of nucleic acid amplification or reverse transcriptase (to prepare cDNA) of for example mRNA in the sample, e.g., by RT-PCR (the experimental embodiment set forth in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi et al. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardi et al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers. In particular aspects of the disclosure, the level of expression of MUC5B is determined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™ System), by a Dual-Glo® Luciferase assay, or by other art-recognized method for measurement of MUC5B expression or mRNA level.

The expression level of MUC5B mRNA may be monitored using a membrane blot (such as used in hybridization analysis such as northern, Southern, dot, and the like), or microwells, sample tubes, gels, beads or fibers (or any solid support comprising bound nucleic acids). See U.S. Pat. Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which are incorporated herein by reference. The determination of MUC5B expression level may also comprise using nucleic acid probes in solution.

In some embodiments, the level of RNA expression is assessed using branched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCR method is described and exemplified in the Examples presented herein. Such methods can also be used for the detection of MUC5B nucleic acids.

The level of MUC5B protein expression may be determined using any method known in the art for the measurement of protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions, absorption spectroscopy, a colorimetric assays, spectrophotometric assays, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assays (ELIS As), immunofluorescent assays, electrochemiluminescence assays, and the like. Such assays can also be used for the detection of proteins indicative of the presence or replication of MUC5B proteins.

In some embodiments, the efficacy of the methods of the disclosure in the treatment of a MUC5B-related disease is assessed by a decrease in MUC5B mRNA level (e.g, by assessment of a MUC5B level, e.g., in the lung, by biopsy, or otherwise, e.g., sputum sample or nasal swab).

In some embodiments, the efficacy of the methods of the disclosure in the treatment of a MUC5B-related disease is assessed by a decrease in MUC5B mRNA level (e.g, by assessment of a liver sample for MUC5B level, by biopsy, or otherwise).

In some embodiments of the methods of the disclosure, the RNAi agent is administered to a subject such that the RNAi agent is delivered to a specific site within the subject. The inhibition of expression of MUC5B may be assessed using measurements of the level or change in the level of MUC5B mRNA or MUC5B protein in a sample derived from a specific site within the subject, e.g., lung and/or liver cells or fluid sample from the respiratory system. In certain embodiments, the methods include a clinically relevant inhibition of expression of MUC5B, e.g. as demonstrated by a clinically relevant outcome after treatment of a subject with an agent to reduce the expression of MUC5B.

As used herein, the terms detecting or determining a level of an analyte are understood to mean performing the steps to determine if a material, e.g., protein, RNA, is present. As used herein, methods of detecting or determining include detection or determination of an analyte level that is below the level of detection for the method used.

IX. Methods of Treating or Preventing MUC5B-Associated Diseases

The present disclosure also provides methods of using a RNAi agent of the disclosure or a composition containing a RNAi agent of the disclosure to reduce or inhibit MUC5B expression in a cell, such as a cell in the respiratory system. The methods include contacting the cell with a dsRNA of the disclosure and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcripts of a MUC5B gene, thereby inhibiting expression of the MUC5B gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of MUC5B may be determined by determining the mRNA expression level of a MUC5B gene using methods routine to one of ordinary skill in the art, e.g., northern blotting, qRT-PCR; by determining the protein level of a MUC5B protein using methods routine to one of ordinary skill in the art, such as western blotting, immunological techniques.

In the methods of the disclosure the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the disclosure may be any cell that expresses a MUC5B gene, such as a cell in the respiratory system that expresses MUC5B. A cell suitable for use in the methods of the disclosure may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a rat cell, or a mouse cell. In one embodiment, the cell is a human cell, e.g., a human lung cell. In one embodiment, the cell is a human cell, e.g., a human liver cell. In one embodiment, the cell is a human cell, e.g., a human lung cell and a human liver cell. MUC5B expression is inhibited in the cell by at least about 30, 40, 50, 55, 60, 65, 70, 75, 80, 90, 95, 96, 97, 98, 99, or about 100%, i.e., to below the level of detection. In preferred embodiments, MUC5B expression is inhibited by at least 50%.

The in vivo methods of the disclosure may include administering to a subject a composition containing a RNAi agent, where the RNAi agent includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the MUC5B gene of the subject to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection. In certain embodiments, the compositions are administered by pulmonary delivery, e.g., oral inhalation or intranasal delivery.

In some embodiments, the administration is via a depot injection. A depot injection may release the RNAi agent in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of MUC5B, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In one embodiment, the double-stranded RNAi agent is administered by pulmonary system administration, e.g., intranasal administration or oral inhalative administration. Pulmonary system administration may be via a syringe, a dropper, atomization, or use of device, e.g., a passive breath driven or active power driven single/-multiple dose dry powder inhaler (DPI) device.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present disclosure also provides methods for inhibiting the expression of a MUC5B gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets a MUC5B gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the RNA transcript of the MUC5B gene, thereby inhibiting expression of the MUC5B gene in the cell. Reduction in genome expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a lung biopsy sample serves as the tissue material for monitoring the reduction in MUC5B gene or protein expression (or of a proxy therefore).

The present disclosure further provides methods of treatment of a subject in need thereof. The treatment methods of the disclosure include administering an RNAi agent of the disclosure to a subject, e.g., a subject that would benefit from inhibition of MUC5B expression, in a therapeutically effective amount of a RNAi agent targeting a MUC5B gene or a pharmaceutical composition comprising a RNAi agent targeting a MUC5B gene.

In addition, the present disclosure provides methods of preventing, treating or inhibiting the progression of a MUC5B-associated disease or disorder, e.g., a lung disease, e.g., cystic fibrosis, chronic obstructive pulmonary disease (COPD), and/or pulmonary fibrosis, e.g., idiopathic pulmonary fibrosis (IPF).

The methods include administering to the subject a therapeutically effective amount of any of the RNAi agent, e.g., dsRNA agents, or the pharmaceutical composition provided herein, thereby preventing, treating, or inhibiting the progression of the MUC5B-associated disease or disorder in the subject, such as IPF.

An RNAi agent of the disclosure may be administered as a “free RNAi agent.” A free RNAi agent is administered in the absence of a pharmaceutical composition. The naked RNAi agent may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the RNAi agent can be adjusted such that it is suitable for administering to a subject. In certain embodiments, the free RNAi agent may be formulated in water or normal saline.

Alternatively, an RNAi agent of the disclosure may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction or inhibition of MUC5B gene expression are those having a MUC5B-associated disease, subjects at risk of developing a MUC5B-associate disease.

The disclosure further provides methods for the use of a RNAi agent or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction or inhibition of MUC5B expression, e.g., a subject having a MUC5B-associated disorder, in combination with other pharmaceuticals or other therapeutic methods, e.g., with known pharmaceuticals or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an RNAi agent targeting MUC5B is administered in combination with, e.g., an agent useful in treating a MUC5B-associated disorder as described elsewhere herein or as otherwise known in the art. For example, additional agents and treatments suitable for treating a subject that would benefit from reduction in MUC5B expression, e.g., a subject having a MUC5B-associated disorder, may include agents currently used to treat symptoms of MUC5B-associated disorder. The RNAi agent and additional therapeutic agents may be administered at the same time or in the same combination, e.g., via pulmonary system administration, or the additional therapeutic agent can be administered as part of a separate composition or at separate times or by another method known in the art or described herein.

Exemplary additional therapeutics and treatments include, for example, an anti-inflammatory agent (e.g., a systemic corticosteroid (e.g., prednisone), an immune modulator (e.g., an immunosuppressant agents (e.g., azathioprine, cyclophosphamide), a phosphodiesterase-5 inhibitor, a tyrosine kinase inhibitor (e.g., nintedanib), an antifibrotic agent (e.g., pirfenidone), and a combination of any of the foregoing.

In one embodiment, the method includes administering a composition featured herein such that expression of the target MUC5B gene is decreased, for at least one month. In preferred embodiments, expression is decreased for at least 2 months, 3 months, or 6 months.

In certain embodiments, administration includes a loading dose administered at a higher frequency, e.g., once per day, twice per week, once per week, for an initial dosing period, e.g., 2-4 doses.

Preferably, the RNAi agents useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target MUC5B gene. Compositions and methods for inhibiting the expression of these genes using RNAi agents can be prepared and performed as described herein.

Administration of the dsRNA according to the methods of the disclosure may result in a reduction of the severity, signs, symptoms, or markers of such diseases or disorders in a patient with a MUC5B-associated disorder. By “reduction” in this context is meant a statistically significant or clinically significant decrease in such level. The reduction can be, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of a RNAi agent targeting MUC5B or pharmaceutical composition thereof, “effective against” a MUC5B-associated disorder indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating MUC5B-associated disorders and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50%, or more can be indicative of effective treatment. Efficacy for a given RNAi agent drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale. Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using a RNAi agent or RNAi agent formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 200 mg/kg.

The RNAi agent can be administered via the pulmonary system over a period of time, on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. Administration of the RNAi agent can reduce MUC5B levels, e.g., in a cell, tissue, blood, lung sample or other compartment of the patient by at least 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70,% 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or at least about 99% or more. In a preferred embodiment, administration of the RNAi agent can reduce MUC5B levels, e.g., in a cell, tissue, blood, pulmonary system sample or other compartment of the patient by at least 50%.

Before administration of a full dose of the RNAi agent, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the RNAi agent can be administered by pulmonary administration or subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired, e.g., monthly dose of RNAi agent to a subject. The injections may be repeated over a period of time. The administration may be repeated on a regular basis. In certain embodiments, after an initial treatment regimen, the treatments can be administered on a less frequent basis. A repeat-dose regimine may include administration of a therapeutic amount of RNAi agent on a regular basis, such as monthly or extending to once a quarter, twice per year, once per year. In certain embodiments, the RNAi agent is administered about once per month to about once per quarter (i.e., about once every three months).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the RNAi agents and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

An informal Sequence Listing is filed herewith and forms part of the specification as filed.

This invention is further illustrated by the following examples which should not be construed as limiting. The entire contents of all references, patents and published patent applications cited throughout this application, as well as the Figures and the Sequence Listing, are hereby incorporated herein by reference.

EXAMPLES Example 1. iRNA Synthesis Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

siRNA Design

The selection of siRNA designs targeting human transmembrane serine protease (MUC5B) gene (human NCBI refseqID: NM_002458.3; NCBI GeneID: 727897) were designed using custo R and Python scripts. The human NM_002458.3 REFSEQ mRNA has a length of 17911 bases.

A detailed list of a set of the unmodified siRNA sense and antisense strand sequences targeting MUC5B is shown in Tables 2, 4, and 6.

A detailed list of a set of the modified siRNA sense and antisense strand sequences targeting MUC5B is shown in Tables 3, 5, and 7.

It is to be understood that, throughout the application, a duplex name without a decimal is equivalent to a duplex name with a decimal which merely references the batch number of the duplex. For example, AD-1230521 is equivalent to AD-1230521.

siRNA Synthesis

siRNAs were synthesized and annealed using routine methods known in the art. Briefly, siRNA sequences were synthesized on a 1 μmol scale using a Mermade 192 synthesizer (BioAutomation) with phosphoramidite chemistry on solid supports. The solid support was controlled pore glass (500-1000 Å) loaded with a custom GalNAc ligand (3′-GalNAc conjugates), universal solid support (AM Chemicals), or the first nucleotide of interest. Ancillary synthesis reagents and standard 2-cyanoethyl phosphoramidite monomers (2′-deoxy-2′-fluoro, 2′-O-methyl, RNA, DNA) were obtained from Thermo-Fisher (Milwaukee, WI), Hongene (China), or Chemgenes (Wilmington, MA, USA). Additional phosphoramidite monomers were procured from commercial suppliers, prepared in-house, or procured using custom synthesis from various CMOs. Phosphoramidites were prepared at a concentration of 100 mM in either acetonitrile or 9:1 acetonitrile:DMF and were coupled using 5-Ethylthio-1H-tetrazole (ETT, 0.25 M in acetonitrile) with a reaction time of 400 s. Phosphorothioate linkages were generated using a 100 mM solution of 3-((Dimethylamino-methylidene) amino)-3H-1,2,4-dithiazole-3-thione (DDTT, obtained from Chemgenes (Wilmington, MA, USA)) in anhydrous acetonitrile/pyridine (9:1 v/v). Oxidation time was 5 minutes. All sequences were synthesized with final removal of the DMT group (“DMT-Off”).

Upon completion of the solid phase synthesis, solid-supported oligoribonucleotides were treated with 300 μL of Methylamine (40% aqueous) at room temperature in 96 well plates for approximately 2 hours to afford cleavage from the solid support and subsequent removal of all additional base-labile protecting groups. For sequences containing any natural ribonucleotide linkages (2′-OH) protected with a tert-butyl dimethyl silyl (TBDMS) group, a second deprotection step was performed using TEA.3HF (triethylamine trihydrofluoride). To each oligonucleotide solution in aqueous methylamine was added 200 μL of dimethyl sulfoxide (DMSO) and 300 μL TEA.3HF and the solution was incubated for approximately 30 mins at 60° C. After incubation, the plate was allowed to come to room temperature and crude oligonucleotides were precipitated by the addition of 1 mL of 9:1 acetontrile:ethanol or 1:1 ethanol:isopropanol. The plates were then centrifuged at 4° C. for 45 mins and the supernatant carefully decanted with the aid of a multichannel pipette. The oligonucleotide pellet was resuspended in 20 mM NaOAc and subsequently desalted using a HiTrap size exclusion column (5 mL, GE Healthcare) on an Agilent LC system equipped with an autosampler, UV detector, conductivity meter, and fraction collector. Desalted samples were collected in 96 well plates and then analyzed by LC-MS and UV spectrometry to confirm identity and quantify the amount of material, respectively.

Duplexing of single strands was performed on a Tecan liquid handling robot. Sense and antisense single strands were combined in an equimolar ratio to a final concentration of 10 μM in 1×PBS in 96 well plates, the plate sealed, incubated at 100° C. for 10 minutes, and subsequently allowed to return slowly to room temperature over a period of 2-3 hours. The concentration and identity of each duplex was confirmed and then subsequently utilized for in vitro screening assays.

Example 2. In Vitro Screening of siRNA Duplexes

Cell Culture and Transfections

Pulmonary system cells, A549 cells (adenocarcinomic human alveolar basal epithelial cells) or MLE12 (murine lung epithelial cells), were cultured according to standard methods and were transfected with the iRNA duplex of interest.

Briefly, cells were transfected by adding 7.5 μL of Opti-MEM plus 0.1 μL of RNAiMAX per well (Invitrogen, Carlsbad CA. cat #13778-150) to 2.5 μL of each siRNA duplex to an individual well in a 384-well plate. The cells were then incubated at room temperature for 15 minutes. Forty tit of MEDIA containing ˜1.5×10⁴ cells was then added to the siRNA mixture. Cells were incubated for 24 hours prior to RNA purification. Single dose experiments were performed in A549 cells at 10 nM. Single dose experiments were performed in MLE12 cells at 10 nM, 1 nM, or 0.1 nM.

In Vitro Dual-Luciferase and Endogenous Screening Assays

Cos7 cells were transfected by adding 50 μL of siRNA duplexes and 75 ng of human MUC5B plasmid per well along with 100 μL of Opti-MEM plus 0.5 μL of Lipofectamine 2000 per well (Invitrogen, Carlsbad CA. cat #13778-150) and then incubated at room temperature for 15 minutes. The mixture was then added to the cells which are re-suspended in 35 μL of fresh complete media. The transfected cells were incubated at 37° C. in an atmosphere of 5% CO2. Single-dose experiments were performed at 10 nM.

Twenty-four hours after the siRNAs and psiCHECK2 plasmid are transfected; Firefly (transfection control) and Renilla (fused to MUC5B target sequence) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 75 μL of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mix. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 75 μL of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 10-15 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenches the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (MUC5B) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done with n=4.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit

Total RNA isolation was performed using DYNABEADS. Briefly, cells were lysed in 10 μl of Lysis/Binding Buffer containing 3 μL of beads per well were mixed for 10 minutes on an electrostatic shaker. The washing steps were automated on a Biotek EL406, using a magnetic plate support. Beads were washed (in 31 μL) once in Buffer A, once in Buffer B, and twice in Buffer E, with aspiration steps in between. Following a final aspiration, complete 12 μL RT mixture was added to each well, as described below.

cDNA Synthesis

For cDNA synthesis, a master mix of 1.5 μl 10× Buffer, 0.6 μl 10× dNTPs, 1.5 μl Random primers, 0.75 μl Reverse Transcriptase, 0.75 μl RNase inhibitor and 9.9 μl of H₂O per reaction were added per well. Plates were sealed, agitated for 10 minutes on an electrostatic shaker, and then incubated at 37 degrees C. for 2 hours. Following this, the plates were agitated at 80 degrees C. for 8 minutes.

Real Time PCR

Two microlitre (μ1) of cDNA were added to a master mix containing 0.5 μl of human GAPDH TaqMan Probe (4326317E), 0.5 μl human MUC5B, 2 μl nuclease-free water and 5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well plates (Roche cat #04887301001). Real time PCR was done in a LightCycler480 Real Time PCR system (Roche).

To calculate relative fold change, data were analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s are calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or mock-transfected. The sense and antisense sequences of AD-1955 are: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO:15) and antisense

(SEQ ID NO: 16) UCGAAGuACUcAGCGuAAGdTsdT.

The results of a single dose in vitro screen of the agents in Tables 4 and 5 in A549 cells are provided in Table 8.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbre- viation Nucleotide(s) A Adenosine-3′-phosphate Ab beta-L-adenosine-3′-phosphate Abs beta-L-adenosine-3′-phosphorothioate Af 2′-fluoroadenosine-3′-phosphate Afs 2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioate C cytidine-3′-phosphate Cb beta-L-cytidine-3′-phosphate Cbs beta-L-cytidine-3'-phosphorothioate Cf 2′-fluorocytidine-3′-phosphate Cfs 2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate G guanosine-3′-phosphate Gb beta-L-guanosine-3′-phosphate Gbs beta-L-guanosine-3′-phosphorothioate Gf 2′-fluoroguanosine-3′-phosphate Gfs 2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioate T 5′-methyluridine-3′-phosphate Tf 2′-fluoro-5-methyluridine-3′-phosphate Tfs 2′-fluoro-5-methyluridine-3′-phosphorothioate Ts 5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs 2′-fluorouridine-3′-phosphorothioate Us uridine-3′-phosphorothioate N any nucleotide, modified or unmodified a 2′-O-methyladenosine-3′-phosphate as 2′-O-methyladenosine-3′-phosphorothioate c 2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs 2′-O-methylguanosine-3′-phosphorothioate t 2′-O-methyl-5-methyluridine-3′-phosphate ts 2′-O-methyl-5-methyluridine-3′-phosphorothioate u 2′-O-methyluridine-3′-phosphate us 2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L10 N-(cholesterylcarboxamidocaproyl)-4-hydroxyprolinol (Hyp-C6-Chol) L96 N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinol (Hyp-(GalNAc-alkyl)3)

Y34 2-hydroxymethyl-tetrahydrofurane-4-methoxy-3-phosphate (abasic 2′-OMe furanose) Y44 inverted abasic DNA (2-hydroxymethyl-tetrahydrofurane-5-phosphate) (Agn) Adenosine-glycol nucleic acid (GNA) (Cgn) Cytidine-glycol nucleic acid (GNA) (Ggn) Guanosine-glycol nucleic acid (GNA) (Tgn) Thymidine-glycol nucleic acid (GNA) S-Isomer P Phosphate VP Vinyl-phosphonate dA 2′-deoxyadenosine-3′-phosphate dAs 2′-deoxyadenosine-3′-phosphorothioate dC 2′-deoxycytidine-3′-phosphate dCs 2′-deoxycytidine-3′-phosphorothioate dG 2′-deoxyguanosine-3′-phosphate dGs 2′-deoxyguanosine-3′-phosphorothioate dT 2′-deoxythymidine-3′-phosphate dTs 2′-deoxythymidine-3′-phosphorothioate dU 2′-deoxyuridine dUs 2′-deoxyuridine-3′-phosphorothioate (C2p) cytidine-2′-phosphate (G2p) guanosine-2′-phosphate (U2p) uridine-2′-phosphate (A2p) adenosine-2′-phosphate (Chd) 2′-O-hexadecyl-cytidine-3′-phosphate (Ahd) 2′-O-hexadecyl-adenosine-3′-phosphate (Ghd) 2′-O-hexadecyl-guanosine-3′-phosphate (Uhd) 2′-O-hexadecyl-uridine-3′-phosphate

TABLE 2 Unmodified Sense and Antisense Strand MUC5B dsRNA Sequences SEQ SEQ Range in ID ID Range in Duplex ID Sense Sequence 5′ to 3′ NM_002458.3 NO: Antisense Sequence 5′ to 3′ NO: NM_002458.3 AD-1302944 UUGGCUCUGGCGGCCAUGCUU  92-112 17 AAGCAUGGCCGCCAGAGCCAACA 348  90-112 AD-1302994 CCGAGCUGGGAGAAUGCAGGU 149-169 18 ACCUGCAUUCUCCCAGCUCGGCU 349 147-169 AD-1303025 GCGCGUGAGCUUUGUUCCACU 211-231 19 AGUGGAACAAAGCUCACGCGCCG 350 209-231 AD-1303054 UGGGCGGGUGUGCAGCACCUU 277-297 20 AAGGUGCUGCACACCCGCCCAUU 351 275-297 AD-1303066 CCACUACAAGACCUUCGACGU 307-327 21 ACGUCGAAGGUCUUGUAGUGGAA 352 305-327 AD-1303108 CCUUUGCAACUACGUGUUCUU 349-369 22 AAGAACACGUAGUUGCAAAGGCC 353 347-369 AD-1303139 GAGGACUUCAACGUCCAGCUU 392-412 23 AAGCUGGACGUUGAAGUCCUCGU 354 390-412 AD-1303190 UCACCCGUGUUGUCAUCAAGU 444-464 24 ACUUGAUGACAACACGGGUGACC 355 442-464 AD-1303222 GGCUCCGUCCUCAUCAAUGGU 494-514 25 ACCAUUGAUGAGGACGGAGCCGU 356 492-514 AD-1303251 GCUGCCUUACAGCCGCACUGU 526-546 26 ACAGUGCGGCUGUAAGGCAGCUC 357 524-546 AD-1303275 GACUACAUCAAGGUCAGCAUU 569-589 27 AAUGCUGACCUUGAUGUAGUCCC 358 567-589 AD-1303304 GCUGACAUUCCUGUGGAACGU 598-618 28 ACGUUCCACAGGAAUGUCAGCAC 359 596-618 AD-1303344 GAGCUGGAUCCCAAAUACGCU 638-658 29 AGCGUAUUUGGGAUCCAGCUCCA 360 636-658 AD-1303368 CAGACCUGUGGCCUGUGUGGU 662-682 30 ACCACACAGGCCACAGGUCUGGU 361 660-682 AD-1303389 GCCUUCAACGAGUUCUAUGCU 701-721 31 AGCAUAGAACUCGUUGAAGGCCG 362 699-721 AD-1303422 GAACCUGCAGAAGUUGGAUGU 754-774 32 ACAUCCAACUUCUGCAGGUUCCC 363 752-774 AD-1303457 UGCACGGACGAGGAGGGCAUU 821-841 33 AAUGCCCUCCUCGUCCGUGCAGU 364 819-841 AD-1303507 GUGGACAGCACUGCGUACCUU 890-910 34 AAGGUACGCAGUGCUGUCCACCA 365 888-910 AD-1303537 GCCACCUUUGUGGAAUACUCU 956-976 35 AGAGUAUUCCACAAAGGUGGCAC 366 954-976 AD-1303569 CUGGAGGUGCCCUGAGCUCUU 1012-1032 36 AAGAGCUCAGGGCACCUCCAGUU 367 1010-1032 AD-1303573 CUCAACAUGCAGCACCAGGAU 1049-1069 37 AUCCUGGUGCUGCAUGUUGAGGG 368 1047-1069 AD-1303602 ACCCUGCACGGACACCUGCUU 1078-1098 38 AAGCAGGUGUCCGUGCAGGGUGA 369 1076-1098 AD-1303626 GGACCACUGUGUGGACGGCUU 1126-1146 39 AAGCCGUCCACACAGUGGUCCUC 370 1124-1146 AD-1303646 GCUGGAUGACAUCACGCACUU 1168-1188 40 AAGUGCGUGAUGUCAUCCAGCAC 371 1166-1188 AD-1303679 CACCUCCUUCAACACCACCUU 1249-1269 41 AAGGUGGUGUUGAAGGAGGUGCC 372 1247-1269 AD-1303705 CUAUGGCAGUGCCAGGACCUU 1295-1315 42 AAGGUCCUGGCACUGCCAUAGCC 373 1293-1315 AD-1303746 CCUAUGAUGAGAAACUCUACU 1362-1382 43 AGUAGAGUUUCUCAUCAUAGGUG 374 1360-1382 AD-1303787 UACGUUCUGUCCAAGAAAUGU 1403-1423 44 ACAUUUCUUGGACAGAACGUAGC 375 1401-1423 AD-1303811 GACAGCAGCUUCACCGUGCUU 1427-1447 45 AAGCACGGUGAAGCUGCUGUCGG 376 1425-1447 AD-1303862 AACGAGAACUGCCUGAAAGCU 1478-1498 46 AGCUUUCAGGCAGUUCUCGUUGU 377 1476-1498 AD-1303914 UCCUCAACUCCAUCUACACGU 1560-1580 47 ACGUGUAGAUGGAGUUGAGGAAC 378 1558-1580 AD-1303932 GCCAACAUCACCCUGUUCACU 1598-1618 48 AGUGAACAGGGUGAUGUUGGCUG 379 1596-1618 AD-1303956 UCGAGCUUCUUCAUCGUGGUU 1622-1642 49 AACCACGAUGAAGAAGCUCGAGG 380 1620-1642 AD-1303977 CAGCUGCUGGUGCAGCUGGUU 1661-1681 50 AACCAGCUGCACCAGCAGCUGCA 381 1659-1681 AD-1304001 CUCAUGCAGGUGUUUGUCAGU 1685-1705 51 ACUGACAAACACCUGCAUGAGUG 382 1683-1705 AD-1304039 GUGGGAACUUCAACCAGAACU 1743-1763 52 AGUUCUGGUUGAAGUUCCCACAC 383 1741-1763 AD-1304064 UGACGACUUCACGGCCCUCAU 1768-1788 53 AUGAGGGCCGUGAAGUCGUCAGC 384 1766-1788 AD-1304077 AGCCUUCGCCAACACCUGGAU 1813-1833 54 AUCCAGGUGUUGGCGAAGGCUGC 385 1811-1833 AD-1304119 CCAGGAACAGCUUUGAGGACU 1857-1877 55 AGUCCUCAAAGCUGUUCCUGGCA 386 1855-1877 AD-1304134 GUGGAGAAUGAGAACUACGCU 1892-1912 56 AGCGUAGUUCUCAUUCUCCACAC 387 1890-1912 AD-1304174 UCCCAACAGUGCCUUCUCGCU 1939-1959 57 AGCGAGAAGGCACUGUUGGGAUC 388 1937-1959 AD-1304202 CUUCCACUCGAACUGCAUGUU 1987-2007 58 AACAUGCAGUUCGAGUGGAAGGG 389 1985-2007 AD-1304226 CACCUGCAACUGUGAGCGGAU 2011-2031 59 AUCCGCUCACAGUUGCAGGUGUC 390 2009-2031 AD-1304262 CCUCCUAUGUGCACGCCUGUU 2058-2078 60 AACAGGCGUGCACAUAGGAGGAC 391 2056-2078 AD-1304286 GGGCGUACAGCUCAGCGACUU 2086-2106 61 AAGUCGCUGAGCUGUACGCCCUU 392 2084-2106 AD-1304322 ACCAAGUACAUGCAGAACUGU 2123-2143 62 ACAGUUCUGCAUGUACUUGGUGC 393 2121-2143 AD-1304326 AAGUCCCAGCGCUACGCCUAU 2147-2167 63 AUAGGCGUAGCGCUGGGACUUGG 394 2145-2167 AD-1304352 GGAUGCCUGCCAGCCCACUUU 2173-2193 64 AAAGUGGGCUGGCAGGCAUCCAC 395 2171-2193 AD-1304386 UCACCUGCAGCGUUUCCUUCU 2217-2237 65 AGAAGGAAACGCUGCAGGUGACG 396 2215-2237 AD-1304415 GGGCACCUUCCUCAAUGACGU 2266-2286 66 ACGUCAUUGAGGAAGGUGCCCGC 397 2264-2286 AD-1304454 CUGGCUCCUGGAGAGGUGGUU 2339-2359 67 AACCACCUCUCCAGGAGCCAGCA 398 2337-2359 AD-1304482 CCGUGUGUUCAUGUACGGGUU 2373-2393 68 AACCCGUACAUGAACACACGGCG 399 2371-2393 AD-1304524 CCUCUCUGCAGAAAAGCACAU 2415-2435 69 AUGUGCUUUUCUGCAGAGAGGCU 400 2413-2435 AD-1304546 CCUGGACUGCAGCAACAGCUU 2458-2478 70 AAGCUGUUGCUGCAGUCCAGGUA 401 2456-2478 AD-1304596 GCUGUUUCAGCACACACUGCU 2532-2552 71 AGCAGUGUGUGCUGAAACAGCCC 402 2530-2552 AD-1304624 CUGCAUUGCCGAGGAGGACUU 2602-2622 72 AAGUCCUCCUCGGCAAUGCAGCC 403 2600-2622 AD-1304646 CACCUACAAGCCUGGAGAGAU 2644-2664 73 AUCUCUCCAGGCUUGUAGGUGGC 404 2642-2664 AD-1304676 CGACUGCAACACCUGCACCUU 2674-2694 74 AAGGUGCAGGUGUUGCAGUCGAC 405 2672-2694 AD-1304700 GAACCGGAGGUGGGAGUGCAU 2698-2718 75 AUGCACUCCCACCUCCGGUUCCU 406 2696-2718 AD-1304732 UGGCCACUUCAUCACCUUUGU 2758-2778 76 ACAAAGGUGAUGAAGUGGCCAUC 407 2756-2778 AD-1304756 CGAUCGCUACAGCUUUGAAGU 2782-2802 77 ACUUCAAAGCUGUAGCGAUCGCC 408 2780-2802 AD-1304779 GCUGCGAGUACAUCUUGGCCU 2805-2825 78 AGGCCAAGAUGUACUCGCAGCUG 409 2803-2825 AD-1304818 UCCGCAUCGUCACCGAGAACU 2862-2882 79 AGUUCUCGGUGACGAUGCGGAAG 410 2860-2882 AD-1304850 AAGGCCAUCAAGCUCUUCGUU 2915-2935 80 AACGAAGAGCUUGAUGGCCUUGG 411 2913-2935 AD-1304873 GAGCUACGAGCUGAUCCUCCU 2938-2958 81 AGGAGGAUCAGCUCGUAGCUCUC 412 2936-2958 AD-1304882 GACCUUUAAGGCGGUGGCGAU 2965-2985 82 AUCGCCACCGCCUUAAAGGUCCC 413 2963-2985 AD-1304894 CACCCUACAAGAUACGCUACU 3003-3023 83 AGUAGCGUAUCUUGUAGGGUGGG 414 3001-3023 AD-1304902 AUCUUCCUGGUCAUCGAGACU 3029-3049 84 AGUCUCGAUGACCAGGAAGAUCC 415 3027-3049 AD-1304953 CAGCGUGUUCAUCCGACUGCU 3082-3102 85 AGCAGUCGGAUGAACACGCUGGU 416 3080-3102 AD-1304977 GGACUACAAGGGCAGGGUCUU 3106-3126 86 AAGACCCUGCCCUUGUAGUCCUG 417 3104-3126 AD-1305014 GACAAUGCCAUCAAUGACUUU 3149-3169 87 AAAGUCAUUGAUGGCAUUGUCGU 418 3147-3169 AD-1305043 GCACUGGAGUUUGGGAACAGU 3200-3220 88 ACUGUUCCCAAACUCCAGUGCGU 419 3198-3220 AD-1305082 CAGAAGCAGUGCAGCAUCCUU 3302-3322 89 AAGGAUGCUGCACUGCUUCUGGG 420 3300-3322 AD-1305104 CCAGGUUGACUCCACCAAGUU 3352-3372 90 AACUUGGUGGAGUCAACCUGGGA 421 3350-3372 AD-1305128 CGAGGCCUGCGUGAACGACGU 3376-3396 91 ACGUCGUUCACGCAGGCCUCGUA 422 3374-3396 AD-1305170 ACUGCGAGUGUUUCUGCACGU 3420-3440 92 ACGUGCAGAAACACUCGCAGUCG 423 3418-3440 AD-1305212 UGUGUGUGUCCUGGCGGACUU 3480-3500 93 AAGUCCGCCAGGACACACACAGG 424 3478-3500 AD-1305228 UGUUCUGUGACUUCUACAACU 3516-3536 94 AGUUGUAGAAGUCACAGAACAAG 425 3514-3536 AD-1305240 CUGUGAGUGGCACUACCAGCU 3547-3567 95 AGCUGGUAGUGCCACUCACAGCC 426 3545-3567 AD-1305297 GCUGCUACCCGAAGUGCCCAU 3642-3662 96 AUGGGCACUUCGGGUAGCAGCCU 427 3640-3662 AD-1305321 AGCCCUUCUUCAAUGAGGACU 3669-3689 97 AGUCCUCAUUGAAGAAGGGCUGG 428 3667-3689 AD-1305347 AAGUGCGUGGCCCAGUGUGGU 3695-3715 98 ACCACACUGGGCCACGCACUUCA 429 3693-3715 AD-1305370 CUACGACAAGGACGGAAACUU 3718-3738 99 AAGUUUCCGUCCUUGUCGUAGCA 430 3716-3738 AD-1305393 AUGACGUCGGUGCAAGGGUCU 3741-3761 100 AGACCCUUGCACCGACGUCAUAG 431 3739-3761 AD-1305410 CCAGAGCUGUAACUGCACACU 3778-3798 101 AGUGUGCAGUUACAGCUCUGGCA 432 3776-3798 AD-1305441 CAGUGCGCUCACAGCCUUGAU 3809-3829 102 AUCAAGGCUGUGAGCGCACUGGA 433 3807-3829 AD-1305471 UGCACCUAUGAGGACAGGACU 3839-3859 103 AGUCCUGUCCUCAUAGGUGCAGG 434 3837-3859 AD-1305501 CAGGACGUCAUCUACAACACU 3869-3889 104 AGUGUUGUAGAUGACGUCCUGGU 435 3867-3889 AD-1305536 CGCCUGCUUGAUCGCCAUCUU 3904-3924 105 AAGAUGGCGAUCAAGCAGGCGCC 436 3902-3924 AD-1305570 ACCAUCAUCAGGAAGGCUGUU 3938-3958 106 AACAGCCUUCCUGAUGAUGGUGC 437 3936-3958 AD-1305610 CCACAACGCCAUUCACCUUCU 3978-3998 107 AGAAGGUGAAUGGCGUUGUGGCU 438 3976-3998 AD-1305643 UCCACCGUGUGUGUCCGCGAU 4046-4066 108 AUCGCGGACACACACGGUGGAGA 439 4044-4066 AD-1305672 GUCCAGCUGGUACAAUGGGCU 4078-4098 109 AGCCCAUUGUACCAGCUGGACCA 440 4076-4098 AD-1305701 GACUUUGAGACGUUUGAAAAU 4127-4147 110 AUUUUCAAACGUCUCAAAGUCUC 441 4125-4147 AD-1305731 AGAGGGUACCAGGUAUGCCCU 4157-4177 111 AGGGCAUACCUGGUACCCUCUCU 442 4155-4177 AD-1305754 GCUGGCUGACAUCGAGUGCCU 4180-4200 112 AGGCACUCGAUGUCAGCCAGCAC 443 4178-4200 AD-1305771 CUUCCCGACAUGCCGCUGGAU 4211-4231 113 AUCCAGCGGCAUGUCGGGAAGCU 444 4209-4231 AD-1305801 CAGGUGGACUGUGACCGCAUU 4244-4264 114 AAUGCGGUCACAGUCCACCUGCU 445 4242-4264 AD-1305817 CGCCAACAGCCAACAGAGUCU 4279-4299 115 AGACUCUGUUGGCUGUUGGCGCA 446 4277-4299 AD-1305821 CUCUGUCACGACUACGAGCUU 4304-4324 116 AAGCUCGUAGUCGUGACAGAGCG 447 4302-4324 AD-1305847 UCUCUGCUGCGAAUACGUGCU 4330-4350 117 AGCACGUAUUCGCAGCAGAGAAC 448 4328-4350 AD-1305889 CACGGAGCCUGCUGUGCCUAU 4405-4425 118 AUAGGCACAGCAGGCUCCGUGCU 449 4403-4425 AD-1305898 AGACCACAGCAACCGAAAAGU 4434-4454 119 ACUUUUCGGUUGCUGUGGUCUGG 450 4432-4454 AD-1305930 CUCGCAGACUGGGUCCAGCUU 4501-4521 120 AAGCUGGACCCAGUCUGCGAGGU 451 4499-4521 AD-1305948 GUGGACAGAGUGGUUUGAUGU 4582-4602 121 ACAUCAAACCACUCUGUCCACUG 452 4580-4602 AD-1305958 CAAGUCUGAACAACUUGGAGU 4612-4632 122 ACUCCAAGUUGUUCAGACUUGGG 453 4610-4632 AD-1305966 UUGAGUCCUACGAUAAGAUCU 4638-4658 123 AGAUCUUAUCGUAGGACUCAACG 454 4636-4658 AD-1306008 GCAGCCUAAGGACAUAGAGUU 4684-4704 124 AACUCUAUGUCCUUAGGCUGCUG 455 4682-4704 AD-1306030 CUGGACCCUGGCACAGGUGGU 4726-4746 125 ACCACCUGUGCCAGGGUCCAGUU 456 4724-4746 AD-1306042 GCACUGUGACGUCCACUUCGU 4756-4776 126 ACGAAGUGGACGUCACAGUGCAC 457 4754-4776 AD-1306069 GUGCAGGAACUGGGAGCAGGU 4783-4803 127 ACCUGCUCCCAGUUCCUGCACAC 458 4781-4803 AD-1306099 CAAGAUGUGCUACAACUACAU 4813-4833 128 AUGUAGUUGUAGCACAUCUUGAA 459 4811-4833 AD-1306132 CUGCUGCAGUGACGACCACUU 4846-4866 129 AAGUGGUCGUCACUGCAGCAGAG 460 4844-4866 AD-1306144 CGACCACAGAGCUGGAGACGU 4893-4913 130 ACGUCUCCAGCUCUGUGGUCGGU 461 4891-4913 AD-1306168 ACCCAGGCCCUGUUCUCAACU 4928-4948 131 AGUUGAGAACAGGGCCUGGGUGG 462 4926-4948 AD-1306198 CUCUCAGAAGGACUGACAUCU 5015-5035 132 AGAUGUCAGUCCUUCUGAGAGGG 463 5013-5035 AD-1306200 CAGAUACACAAGCACCCUUGU 5038-5058 133 ACAAGGGUGCUUGUGUAUCUGGG 464 5036-5058 AD-1306235 GCUCCACAGAACCCACUGUCU 5094-5114 134 AGACAGUGGGUUCUGUGGAGCCU 465 5092-5114 AD-1306254 CACCCUUCCAACACGCUCAGU 5131-5151 135 ACUGAGCGUGUUGGAAGGGUGGA 466 5129-5151 AD-1306278 CAACAACAAUGGCAACCUCCU 5214-5234 136 AGGAGGUUGCCAUUGUUGUUGGG 467 5212-5234 AD-1306324 ACCGCUUCCAAAGAGCCGCUU 5261-5281 137 AAGCGGCUCUUUGGAAGCGGUGC 468 5259-5281 AD-1306359 CCAACACUCACGAGCGAGCUU 5297-5317 138 AAGCUCGCUCGUGAGUGUUGGCG 469 5295-5317 AD-1306382 CACCUCUCAGGCCGAGACCAU 5320-5340 139 AUGGUCUCGGCCUGAGAGGUGGA 470 5318-5340 AD-1306406 CCAGGACAGAGACGACAAUGU 5346-5366 140 ACAUUGUCGUCUCUGUCCUGGGC 471 5344-5366 AD-1306411 CUUGACUAACACCACCACCAU 5371-5391 141 AUGGUGGUGGUGUUAGUCAAGGG 472 5369-5391 AD-1306442 CUGUCAACCGAAGUGUGAGUU 5407-5427 142 AACUCACACUUCGGUUGACAGCG 473 5405-5427 AD-1306478 CGUGGACUUCCCAACCUCAGU 5443-5463 143 ACUGAGGUUGGGAAGUCCACGUC 474 5441-5463 AD-1306483 ACAUGGAAACUUUUGAAAACU 5478-5498 144 AGUUUUCAAAAGUUUCCAUGUCC 475 5476-5498 AD-1306514 GCACCAAAGAGCAUAGAGUGU 5528-5548 145 ACACUCUAUGCUCUUUGGUGCCC 476 5526-5548 AD-1306535 GGUAAGCAUCGACCAGGUCGU 5569-5589 146 ACGACCUGGUCGAUGCUUACCUC 477 5567-5589 AD-1306563 CUGACCUGCAGCCUGGAGACU 5597-5617 147 AGUCUCCAGGCUGCAGGUCAGCA 478 5595-5617 AD-1306575 UGCAAGAACGAAGACCAGACU 5627-5647 148 AGUCUGGUCUUCGUUCUUGCAGG 479 5625-5647 AD-1306603 UCAACAUGUGCUUCAACUACU 5655-5675 149 AGUAGUUGAAGCACAUGUUGAAC 480 5653-5675 AD-1306627 UGCGUGUGCUUUGCUGUGACU 5679-5699 150 AGUCACAGCAAAGCACACGCACG 481 5677-5699 AD-1306673 CUCCACCCUGAGAACAGCUCU 5839-5859 151 AGAGCUGUUCUCAGGGUGGAGGU 482 5837-5859 AD-1306676 UCCCAAAGUGCUGACCACCAU 5863-5883 152 AUGGUGGUCAGCACUUUGGGAGG 483 5861-5883 AD-1306698 UUCCUCCCUGGGCACCACCUU 6013-6033 153 AAGGUGGUGCCCAGGGAGGAAGA 484 6011-6033 AD-1306717 UGCCAACUACCACAACCACGU 6210-6230 154 ACGUGGUUGUGGUAGUUGGCACU 485 6208-6230 AD-1307191 CACACCCACAACCAGAGGCUU 6301-6321 155 AAGCCUCUGGUUGUGGGUGUGGU 486 6299-6321 AD-1307354 GACCUGGAUCCUCACAAAGCU 5767-5787 156 AGCUUUGUGAGGAUCCAGGUCGU 487 5765-5787 AD-1308029 CUACCAGCGUUACACCCAUCU 5988-6008 157 AGAUGGGUGUAACGCUGGUAGCU 488 5986-6008 AD-1308183 CCACAACAGCCACUACGACUU 5790-5810 158 AAGUCGUAGUGGCUGUUGUGGUC 489 5788-5810 AD-1308294 CAGCUCCAAAGCCACUCCCUU 5905-5925 159 AAGGGAGUGGCUUUGGAGCUGGU 490 5903-5925 AD-1334092 GCCCUUCCAGCACUGAGAAGU 5948-5968 160 ACUUCUCAGUGCUGGAAGGGCGG 491 5946-5968 AD-1308403 CCUAUCACAGACCACCACACU 6040-6060 161 AGUGUGGUGGUCUGUGAUAGGCG 492 6038-6060 AD-1308420 GGCCACCAUGUCCACAGCCAU 6064-6084 162 AUGGCUGUGGACAUGGUGGCCGU 493 6062-6084 AD-1308440 CCUCCUCCACUCCAGAGACUU 6087-6107 163 AAGUCUCUGGAGUGGAGGAGGGU 494 6085-6107 AD-1334093 CACACCUCCACAGUGCUUACU 6110-6130 164 AGUAAGCACUGUGGAGGUGUGGG 495 6108-6130 AD-1308488 CAGGAACAGCUCACACUACCU 6186-6206 165 AGGUAGUGUGAGCUGUUCCUGGG 496 6184-6206 AD-1308555 UCCAGUGUGGAUCAGCACAAU 6277-6297 166 AUUGUGCUGAUCCACACUGGAGG 497 6275-6297 AD-1306730 CUCCUGGGACAACUCCCAUCU 6513-6533 167 AGAUGGGAGUUGUCCCAGGAGUU 498 6511-6533 AD-1306746 CAGCAACACAGUGACUCCCUU 6574-6594 168 AAGGGAGUCACUGUGUUGCUGGU 499 6572-6594 AD-1306753 AGUGCCGAACACCAUGGCCAU 6625-6645 169 AUGGCCAUGGUGUUCGGCACUGG 500 6623-6645 AD-1306772 GGUGACUUCCCACACCCUAGU 6754-6774 170 ACUAGGGUGUGGGAAGUCACCGU 501 6752-6774 AD-1306796 CGACUCCAGCCCUUUCCAGCU 6801-6821 171 AGCUGGAAAGGGCUGGAGUCGAG 502 6799-6821 AD-1306820 UAGCAGCAGAACCACCGAGUU 6829-6849 172 AACUCGGUGGUUCUGCUGCUAGG 503 6827-6849 AD-1306844 GCUCACACUACCAAAGUGCUU 7865-7885 173 AAGCACUUUGGUAGUGUGAGCUG 504 7863-7885 AD-1306879 ACGCUUCCAGUGUGGAUCAGU 7943-7963 174 ACUGAUCCACACUGGAAGCGUGC 505 7941-7963 AD-1306887 CACCCACAACCAGAGGUUCCU 7974-7994 175 AGGAACCUCUGGUUGUGGGUGUG 506 7972-7994 AD-1306916 CACGGUGGUGACCAUGGGCUU 6979-6999 176 AAGCCCAUGGUCACCACCGUGGU 507 6977-6999 AD-1307191 CACACCCACAACCAGAGGCUU 6301-6321 155 AAGCCUCUGGUUGUGGGUGUGGU 486 6299-6321 AD-1307212 ACCGCCACAGUGCUGACCACU 6359-6379 177 AGUGGUCAGCACUGUGGCGGUGU 508 6357-6379 AD-1307378 CCACUACGACUGAGUCCACUU 7386-7406 178 AAGUGGACUCAGUCGUAGUGGCU 509 7384-7406 AD-1307392 GCUCCAAAGCCACUCCCUUCU 7578-7598 179 AGAAGGGAGUGGCUUUGGAGCUG 510 7576-7598 AD-1307516 CUCCAGGGACAACACCUAUCU 8184-8204 180 AGAUAGGUGUUGUCCCUGGAGUU 511 8182-8204 AD-1307551 CAGCAGCACAGUGACUCCCUU 8245-8265 181 AAGGGAGUCACUGUGCUGCUGGU 512 8243-8265 AD-1307575 UGCCCUAGGGACCACCCACAU 6598-6618 182 AUGUGGGUGGUCCCUAGGGCAGA 513 6596-6618 AD-1307600 CACACACGGGCGAUCCCUGUU 8317-8337 183 AACAGGGAUCGCCCGUGUGUGGU 514 8315-8337 AD-1307617 AGCCUGGACUUCGGCCACCUU 6694-6714 184 AAGGUGGCCGAAGUCCAGGCUGU 515 6692-6714 AD-1307654 ACCCACAUCACAGAGCCUUCU 6731-6751 185 AGAAGGCUCUGUGAUGUGGGUGG 516 6729-6751 AD-1307666 CAACCACCGGUACCACCCAGU 6777-6797 186 ACUGGGUGGUACCGGUGGUUGCU 517 6775-6797 AD-1307754 ACCCAGCAAGACCCGCACCUU 6919-6939 187 AAGGUGCGGGUCUUGCUGGGUGU 518 6917-6939 AD-1307805 GUGGCUGGACUACAGCUACCU 7024-7044 188 AGGUAGCUGUAGUCCAGCCACUC 519 7022-7044 AD-1307812 UUGACACCUACUCCAACAUCU 7071-7091 189 AGAUGUUGGAGUAGGUGUCAAAG 520 7069-7091 AD-1307837 UUGGGCCAGGUCGUGGAAUGU 7175-7195 190 ACAUUCCACGACCUGGCCCAACU 521 7173-7195 AD-1307860 CCUGGACUUUGGCCUGGUCUU 7198-7218 191 AAGACCAGGCCAAAGUCCAGGCU 522 7196-7218 AD-1307893 GAUGUGCUUCAACUAUGAAAU 7249-7269 192 AUUUCAUAGUUGAAGCACAUCUU 523 7247-7269 AD-1307917 UGUGUUCUGCUGCAACUACGU 7273-7293 193 ACGUAGUUGCAGCAGAACACACG 524 7271-7293 AD-1307934 CAGCUCUACGGCCAUGCCCUU 7318-7338 194 AAGGGCAUGGCCGUAGAGCUGGU 525 7316-7338 AD-1308171 AUCCUCACAGAGCUGACCACU 7361-7381 195 AGUGGUCAGCUCUGUGAGGAUCC 526 7359-7381 AD-1308216 CCGAGCACUACAGCCACCGUU 7460-7480 196 AACGGUGGCUGUAGUGCUCGGCU 527 7458-7480 AD-1308250 CCUCCACCCAGGCAACUGCUU 7512-7532 197 AAGCAGUUGCCUGGGUGGAGGAG 528 7510-7532 AD-1308273 GGCCACGACACCCACAGUCAU 7555-7575 198 AUGACUGUGGGUGUCGUGGCCGU 529 7553-7575 AD-1334092 GCCCUUCCAGCACUGAGAAGU 5948-5968 160 ACUUCUCAGUGCUGGAAGGGCGG 491 5946-5968 AD-1308369 CACAGCUACCAGCUUUACAGU 7654-7674 199 ACUGUAAAGCUGGUAGCUGUGGG 530 7652-7674 AD-1308403 CCUAUCACAGACCACCACACU 6040-6060 161 AGUGUGGUGGUCUGUGAUAGGCG 492 6038-6060 AD-1308420 GGCCACCAUGUCCACAGCCAU 6064-6084 162 AUGGCUGUGGACAUGGUGGCCGU 493 6062-6084 AD-1308440 CCUCCUCCACUCCAGAGACUU 6087-6107 163 AAGUCUCUGGAGUGGAGGAGGGU 494 6085-6107 AD-1308463 CACACCUCCACAGUGCUUACU 6110-6130 164 AGUAAGCACUGUGGAGGUGUGGA 531 7779-7801 AD-1308520 ACCACAACCACGGGCUUCACU 6218-6238 200 AGUGAAGCCCGUGGUUGUGGUAG 532 6216-6238 AD-1308605 GACCACCACCACCACAACUGU 6373-6393 201 ACAGUUGUGGUGGUGGUGGUCAG 533 6371-6393 AD-1308629 CACUGGUUCUAUGGCAACACU 6397-6417 202 AGUGUUGCCAUAGAACCAGUGGC 534 6395-6417 AD-1308652 CCUCUAGCACACAGACCAGUU 6420-6440 203 AACUGGUCUGUGUGCUAGAGGAG 535 6418-6440 AD-1308673 CCACGGCCACUACGAUCACGU 6462-6482 204 ACGUGAUCGUAGUGGCCGUGGUG 536 6460-6482 AD-1309037 GGGACCACCUGGAUCCUCACU 7436-7456 205 AGUGAGGAUCCAGGUGGUCCCUG 537 7434-7456 AD-1306916 CACGGUGGUGACCAUGGGCUU 6979-6999 176 AAGCCCAUGGUCACCACCGUGGU 507 6977-6999 AD-1306975 CAGCCACUACGACCGCAACCU 9054-9074 206 AGGUUGCGGUCGUAGUGGCUGCU 538 9052-9074 AD-1307006 UCCCAAAGUGCUGACCAGCAU 9121-9141 207 AUGCUGGUCAGCACUUUGGGAGG 539 9119-9141 AD-1307011 CUCCUUCACCCUUGGGACCAU 9268-9288 208 AUGGUCCCAAGGGUGAAGGAGGG 540 9266-9288 AD-1307040 GGGCCACCAGUUCCAUGUCCU 9408-9428 209 AGGACAUGGAACUGGUGGCCCUU 541 9406-9428 AD-1307063 CAGCACUACAGCCACCGUGAU 9559-9579 210 AUCACGGUGGCUGUAGUGCUGGG 542 9557-9579 AD-1307092 ACCCUCAAAGUGCUGACCAGU 9632-9652 211 ACUGGUCAGCACUUUGAGGGUGC 543 9630-9652 AD-1307111 CACACCCACAGUCAUCAGCUU 9661-9681 212 AAGCUGAUGACUGUGGGUGUGGU 544 9659-9681 AD-1307137 CACCCACAGCUACCAGCGUUU 5979-5999 213 AAACGCUGGUAGCUGUGGGUGUG 545 5977-5999 AD-1307167 UCCUCUACUCCAGAGACUGUU 9860-9880 214 AACAGUCUCUGGAGUAGAGGAGG 546 9858-9880 AD-1307174 ACAGUGCUUACCACCACGACU 9890-9910 215 AGUCGUGGUGGUAAGCACUGUGG 547 9888-9910 AD-1307191 CACACCCACAACCAGAGGCUU 6301-6321 155 AAGCCUCUGGUUGUGGGUGUGGU 486 6299-6321 AD-1307212 ACCGCCACAGUGCUGACCACU 6359-6379 177 AGUGGUCAGCACUGUGGCGGUGU 508 6357-6379 AD-1307222 GCCACUACGAUCACAGCCACU 10238-10258 216 AGUGGCUGUGAUCGUAGUGGCCG 548 10236-10258 AD-1307242 CUCCAGGGACAACUCCCAUCU 10284-10304 217 AGAUGGGAGUUGUCCCUGGAGUU 549 10282-10304 AD-1307288 CAGCAGCAACCACCAGUACCU 10542-10562 218 AGGUACUGGUGGUUGCUGCUGGG 550 10540-10562 AD-1307310 CUCCAGGACCACAGCCACAGU 10666-10686 219 ACUGUGGCUGUGGUCCUGGAGGU 551 10664-10686 AD-1307346 CGGCCACGCCCUCCUCAACUU 5739-5759 220 AAGUUGAGGAGGGCGUGGCCGUA 552 11095-11117 AD-1307363 CCUCACAAAGCUGACCACAAU 11134-11154 221 AUUGUGGUCAGCUUUGUGAGGAU 553 11132-11154 AD-1307378 CCACUACGACUGAGUCCACUU 7386-7406 178 AAGUGGACUCAGUCGUAGUGGCU 509 7384-7406 AD-1307392 GCUCCAAAGCCACUCCCUUCU 7578-7598 179 AGAAGGGAGUGGCUUUGGAGCUG 510 7576-7598 AD-1307431 UCCUCCACUCCAGAGACUGCU 6089-6109 222 AGCAGUCUCUGGAGUGGAGGAGG 554 6087-6109 AD-1307456 CCACAGUGCUUACCACCACGU 7788-7808 223 ACGUGGUGGUAAGCACUGUGGAG 555 7786-7808 AD-1307516 CUCCAGGGACAACACCUAUCU 8184-8204 180 AGAUAGGUGUUGUCCCUGGAGUU 511 8182-8204 AD-1307551 CAGCAGCACAGUGACUCCCUU 8245-8265 181 AAGGGAGUCACUGUGCUGCUGGU 512 8243-8265 AD-1307575 UGCCCUAGGGACCACCCACAU 6598-6618 182 AUGUGGGUGGUCCCUAGGGCAGA 513 6596-6618 AD-1307590 CACCACGGCCACCACACACGU 8305-8325 224 ACGUGUGUGGUGGCCGUGGUGUU 556 8303-8325 AD-1307600 CACACACGGGCGAUCCCUGUU 8317-8337 183 AACAGGGAUCGCCCGUGUGUGGU 514 8315-8337 AD-1307617 AGCCUGGACUUCGGCCACCUU 6694-6714 184 AAGGUGGCCGAAGUCCAGGCUGU 515 6692-6714 AD-1307654 ACCCACAUCACAGAGCCUUCU 6731-6751 185 AGAAGGCUCUGUGAUGUGGGUGG 516 6729-6751 AD-1307677 ACCACCCAGCACUCGACUCCU 6788-6808 225 AGGAGUCGAGUGCUGGGUGGUAC 557 6786-6808 AD-1307706 CAGCCCUCACCCUAGCAGCAU 6817-6837 226 AUGCUGCUAGGGUGAGGGCUGGA 558 6815-6837 AD-1307754 ACCCAGCAAGACCCGCACCUU 6919-6939 187 AAGGUGCGGGUCUUGCUGGGUGU 518 6917-6939 AD-1307775 AUAACCACGGUGGUGACCACU 10745-10765 227 AGUGGUCACCACCGUGGUUAUGG 559 10743-10765 AD-1307805 GUGGCUGGACUACAGCUACCU 7024-7044 188 AGGUAGCUGUAGUCCAGCCACUC 519 7022-7044 AD-1307812 UUGACACCUACUCCAACAUCU 7071-7091 189 AGAUGUUGGAGUAGGUGUCAAAG 520 7069-7091 AD-1307837 UUGGGCCAGGUCGUGGAAUGU 7175-7195 190 ACAUUCCACGACCUGGCCCAACU 521 7173-7195 AD-1307860 CCUGGACUUUGGCCUGGUCUU 7198-7218 191 AAGACCAGGCCAAAGUCCAGGCU 522 7196-7218 AD-1307893 GAUGUGCUUCAACUAUGAAAU 7249-7269 192 AUUUCAUAGUUGAAGCACAUCUU 523 7247-7269 AD-1307917 UGUGUUCUGCUGCAACUACGU 7273-7293 193 ACGUAGUUGCAGCAGAACACACG 524 7271-7293 AD-1307934 CAGCUCUACGGCCAUGCCCUU 7318-7338 194 AAGGGCAUGGCCGUAGAGCUGGU 525 7316-7338 AD-1307994 CCAGUUCCAAAGCCACUUCCU 9162-9182 228 AGGAAGUGGCUUUGGAACUGGUG 560 9160-9182 AD-1308057 CCCAGAACAGACCACCACACU 9298-9318 229 AGUGUGGUGGUCUGUUCUGGGAG 561 9296-9318 AD-1308098 CUCCACAGUGCUGACCACGAU 9373-9393 230 AUCGUGGUCAGCACUGUGGAGGU 562 9371-9393 AD-1308142 GACCUGGAUCCUCACAGAGCU 7354-7374 231 AGCUCUGUGAGGAUCCAGGUCGU 563 7352-7374 AD-1308171 AUCCUCACAGAGCUGACCACU 7361-7381 195 AGUGGUCAGCUCUGUGAGGAUCC 526 7359-7381 AD-1308216 CCGAGCACUACAGCCACCGUU 7460-7480 196 AACGGUGGCUGUAGUGCUCGGCU 527 7458-7480 AD-1308250 CCUCCACCCAGGCAACUGCUU 7512-7532 197 AAGCAGUUGCCUGGGUGGAGGAG 528 7510-7532 AD-1308273 GGCCACGACACCCACAGUCAU 7555-7575 198 AUGACUGUGGGUGUCGUGGCCGU 529 7553-7575 AD-1308305 CCACUCCCUCCUCCAGUCCAU 5916-5936 232 AUGGACUGGAGGAGGGAGUGGCU 564 5914-5936 AD-1334092 GCCCUUCCAGCACUGAGAAGU 5948-5968 160 ACUUCUCAGUGCUGGAAGGGCGG 491 5946-5968 AD-1308369 CACAGCUACCAGCUUUACAGU 7654-7674 199 ACUGUAAAGCUGGUAGCUGUGGG 530 7652-7674 AD-1308403 CCUAUCACAGACCACCACACU 6040-6060 161 AGUGUGGUGGUCUGUGAUAGGCG 492 6038-6060 AD-1308422 CCACCAUGUCCACAGCCACAU 6066-6086 233 AUGUGGCUGUGGACAUGGUGGCC 565 6064-6086 AD-1308488 CAGGAACAGCUCACACUACCU 6186-6206 165 AGGUAGUGUGAGCUGUUCCUGGG 496 6184-6206 AD-1308512 UGCCGACUACCACAACCACGU  9981-10001 234 ACGUGGUUGUGGUAGUCGGCACU 566 9979-10001 AD-1308555 UCCAGUGUGGAUCAGCACAAU 6277-6297 166 AUUGUGCUGAUCCACACUGGAGG 497 6275-6297 AD-1308569 CACACCCACAACCAGUGGCUU 11743-11763 235 AAGCCACUGGUUGUGGGUGUGGU 567 11741-11763 AD-1308605 GACCACCACCACCACAACUGU 6373-6393 201 ACAGUUGUGGUGGUGGUGGUCAG 533 6371-6393 AD-1308629 CACUGGUUCUAUGGCAACACU 6397-6417 202 AGUGUUGCCAUAGAACCAGUGGC 534 6395-6417 AD-1308652 CCUCUAGCACACAGACCAGUU 6420-6440 203 AACUGGUCUGUGUGCUAGAGGAG 535 6418-6440 AD-1308673 CCACGGCCACUACGAUCACGU 6462-6482 204 ACGUGAUCGUAGUGGCCGUGGUG 536 6460-6482 AD-1308759 CAAGGACUGCAACCACCCUUU 9192-9212 236 AAAGGGUGGUUGCAGUCCUUGGA 568 9190-9212 AD-1308782 GUGCUGACAAGCACAGCCACU 9215-9235 237 AGUGGCUGUGCUUGUCAGCACUG 569 9213-9235 AD-1308808 CACAGCUACCAGCUUUACACU 9241-9261 238 AGUGUAAAGCUGGUAGCUGUGGA 570 9239-9261 AD-1334094 CCACCAUGUCCACAAUCCACU 9324-9344 239 AGUGGAUUGUGGACAUGGUGGCC 571 9322-9344 AD-1309007 CCACUACAACUGCAGCCACUU 9483-9503 240 AAGUGGCUGCAGUUGUAGUGGCU 572 9481-9503 AD-1309037 GGGACCACCUGGAUCCUCACU 7436-7456 205 AGUGAGGAUCCAGGUGGUCCCUG 537 7434-7456 AD-1309039 GACCACCUGGAUCCUCACAGU 7438-7458 241 ACUGUGAGGAUCCAGGUGGUCCC 573 7436-7458 AD-1307953 CCACUACGACUGCAUCCACUU 12828-12848 242 AAGUGGAUGCAGUCGUAGUGGCU 574 12826-12848 AD-1307967 CUCCCAAAGUGCUGACCAGCU 9120-9140 243 AGCUGGUCAGCACUUUGGGAGGG 575 9118-9140 AD-1307994 CCAGUUCCAAAGCCACUUCCU 9162-9182 228 AGGAAGUGGCUUUGGAACUGGUG 560 9160-9182 AD-1308024 CACAGCUACCAGCGUUACACU 5983-6003 244 AGUGUAACGCUGGUAGCUGUGGA 576 13010-13032 AD-1308057 CCCAGAACAGACCACCACACU 9298-9318 229 AGUGUGGUGGUCUGUUCUGGGAG 561 9296-9318 AD-1308098 CUCCACAGUGCUGACCACGAU 9373-9393 230 AUCGUGGUCAGCACUGUGGAGGU 562 9371-9393 AD-1308133 GGGCCACCAGUUCCACGUCCU 13179-13199 245 AGGACGUGGAACUGGUGGCCCUU 577 13177-13199 AD-1308171 AUCCUCACAGAGCUGACCACU 7361-7381 195 AGUGGUCAGCUCUGUGAGGAUCC 526 7359-7381 AD-1308180 UGACCACAACAGCCACUACGU 7374-7394 246 ACGUAGUGGCUGUUGUGGUCAGC 578 7372-7394 AD-1308216 CCGAGCACUACAGCCACCGUU 7460-7480 196 AACGGUGGCUGUAGUGCUCGGCU 527 7458-7480 AD-1308250 CCUCCACCCAGGCAACUGCUU 7512-7532 197 AAGCAGUUGCCUGGGUGGAGGAG 528 7510-7532 AD-1308263 UGAGCACCACGGCCACGACAU 7545-7565 247 AUGUCGUGGCCGUGGUGCUCACA 579 7543-7565 AD-1308294 CAGCUCCAAAGCCACUCCCUU 5905-5925 159 AAGGGAGUGGCUUUGGAGCUGGU 490 5903-5925 AD-1308317 CCAGUCCAGGGACUGCAACUU 13554-13574 248 AAGUUGCAGUCCCUGGACUGGAG 580 13552-13574 AD-1308344 CAGCACUGAGAAGCACAGCCU 5955-5975 249 AGGCUGUGCUUCUCAGUGCUGGA 581 5953-5975 AD-1308369 CACAGCUACCAGCUUUACAGU 7654-7674 199 ACUGUAAAGCUGGUAGCUGUGGG 530 7652-7674 AD-1308403 CCUAUCACAGACCACCACACU 6040-6060 161 AGUGUGGUGGUCUGUGAUAGGCG 492 6038-6060 AD-1308420 GGCCACCAUGUCCACAGCCAU 6064-6084 162 AUGGCUGUGGACAUGGUGGCCGU 493 6062-6084 AD-1308440 CCUCCUCCACUCCAGAGACUU 6087-6107 163 AAGUCUCUGGAGUGGAGGAGGGU 494 6085-6107 AD-1308463 CACACCUCCACAGUGCUUACU 6110-6130 164 AGUAAGCACUGUGGAGGUGUGGA 531 7779-7801 AD-1308488 CAGGAACAGCUCACACUACCU 6186-6206 165 AGGUAGUGUGAGCUGUUCCUGGG 496 6184-6206 AD-1308512 UGCCGACUACCACAACCACGU 9981-10001 234 ACGUGGUUGUGGUAGUCGGCACU 566 9979-10001 AD-1308555 UCCAGUGUGGAUCAGCACAAU 6277-6297 166 AUUGUGCUGAUCCACACUGGAGG 497 6275-6297 AD-1308566 CACCACACCCACAACCAGUGU 11740-11760 250 ACACUGGUUGUGGGUGUGGUGGU 582 11738-11760 AD-1308595 CGCCAGAGUGCUGACCACCAU 14002-14022 251 AUGGUGGUCAGCACUCUGGCGGU 583 14000-14022 AD-1308629 CACUGGUUCUAUGGCAACACU 6397-6417 202 AGUGUUGCCAUAGAACCAGUGGC 534 6395-6417 AD-1308652 CCUCUAGCACACAGACCAGUU 6420-6440 203 AACUGGUCUGUGUGCUAGAGGAG 535 6418-6440 AD-1308673 CCACGGCCACUACGAUCACGU 6462-6482 204 ACGUGAUCGUAGUGGCCGUGGUG 536 6460-6482 AD-1308702 CAGGGACAACACCCAUCACCU 14157-14177 252 AGGUGAUGGGUGUUGUCCCUGGA 584 14155-14177 AD-1308733 CUCCAAAGCCACUUCCUCCUU 14218-14238 253 AAGGAGGAAGUGGCUUUGGAGCU 585 14216-14238 AD-1308759 CAAGGACUGCAACCACCCUUU 9192-9212 236 AAAGGGUGGUUGCAGUCCUUGGA 568 9190-9212 AD-1308782 GUGCUGACAAGCACAGCCACU 9215-9235 237 AGUGGCUGUGCUUGUCAGCACUG 569 9213-9235 AD-1308783 UGCUGACAAGCACAGCCACAU 14268-14288 254 AUGUGGCUGUGCUUGUCAGCACU 586 14266-14288 AD-1308808 CACAGCUACCAGCUUUACACU 9241-9261 238 AGUGUAAAGCUGGUAGCUGUGGA 570 9239-9261 AD-1308818 CUCCACCCUGUGGACCACGUU 14323-14343 255 AACGUGGUCCACAGGGUGGAGGA 587 14321-14343 AD-1308845 CCCAGCACAGACCACCACACU 14350-14370 256 AGUGUGGUGGUCUGUGCUGGGAC 588 14348-14370 AD-1308868 GUCCACCAUGUCCACAAUCCU 14374-14394 257 AGGAUUGUGGACAUGGUGGACAU 589 14372-14394 AD-1334094 CCACCAUGUCCACAAUCCACU 9324-9344 239 AGUGGAUUGUGGACAUGGUGGCC 571 9322-9344 AD-1308890 CCUCCUCUACUCCAGAGACCU 14397-14417 258 AGGUCUCUGGAGUAGAGGAGGUG 590 14395-14417 AD-1308912 CACACCUCCACAGUGCUGACU 9368-9388 259 AGUCAGCACUGUGGAGGUGUGGG 591 9366-9388 AD-1308935 CACAGCCACCAUGACAAGGGU 14443-14463 260 ACCCUUGUCAUGGUGGCUGUGGU 592 14441-14463 AD-1308963 UCCACGGCCACACCCUCCUCU 14471-14491 261 AGAGGAGGGUGUGGCCGUGGAAU 593 14469-14491 AD-1308975 GACCCGGAUCCUCACUGAGCU 14503-14523 262 AGCUCAGUGAGGAUCCGGGUCGU 594 14501-14523 AD-1308998 CCACAACAGCCACUACAACUU 14526-14546 263 AAGUUGUAGUGGCUGUUGUGGUC 595 14524-14546 AD-1309007 CCACUACAACUGCAGCCACUU 9483-9503 240 AAGUGGCUGCAGUUGUAGUGGCU 572 9481-9503 AD-1309026 UGGAUCCACGGCCACCCUGUU 14554-14574 264 AACAGGGUGGCCGUGGAUCCAGU 596 14552-14574 AD-1309037 GGGACCACCUGGAUCCUCACU 7436-7456 205 AGUGAGGAUCCAGGUGGUCCCUG 537 7434-7456 AD-1309039 GACCACCUGGAUCCUCACAGU 7438-7458 241 ACUGUGAGGAUCCAGGUGGUCCC 573 7436-7458 AD-1309042 CACCUGGAUCCUCACAGAGCU 7441-7461 265 AGCUCUGUGAGGAUCCAGGUGGU 597 7439-7461 AD-1309069 UAUAGCCACCGUGAUGGUGCU 14617-14637 266 AGCACCAUCACGGUGGCUAUAGU 598 14615-14637 AD-1309105 CCACUCUGGGAACAGCUCACU 14664-14684 267 AGUGAGCUGUUCCCAGAGUGGAG 599 14662-14684 AD-1309124 CAUGGCCACUAUGCCCACAGU 14704-14724 268 ACUGUGGGCAUAGUGGCCAUGGU 600 14702-14724 AD-1309148 UGCCUCCACGGUUCCCAGCUU 14728-14748 269 AAGCUGGGAACCGUGGAGGCAGU 601 14726-14748 AD-1309167 CCUGCCAACCUUCAGCGUGUU 14794-14814 270 AACACGCUGAAGGUUGGCAGGCU 602 14792-14814 AD-1309192 GUGUCCUCCUCAGUCCUCACU 14819-14839 271 AGUGAGGACUGAGGAGGACACAG 603 14817-14839 AD-1309216 CAGCUCCCACUUCUCUACUCU 14863-14883 272 AGAGUAGAGAAGUGGGAGCUGGG 604 14861-14883 AD-1309250 GCAUUUGGACAGUUUUUCUCU 14897-14917 273 AGAGAAAAACUGUCCAAAUGCCC 605 14895-14917 AD-1309259 GAAGUCAUCUACAAUAAGACU 14924-14944 274 AGUCUUAUUGUAGAUGACUUCCC 606 14922-14944 AD-1309287 CUGCCAUUUCUACGCAGUGUU 14956-14976 275 AACACUGCGUAGAAAUGGCAGCC 607 14954-14976 AD-1309315 CACUGUGACAUUGACCGCUUU 14984-15004 276 AAAGCGGUCAAUGUCACAGUGCU 608 14982-15004 AD-1309337 UGUGACAAUGCCAUCCCUCUU 15077-15097 277 AAGAGGGAUGGCAUUGUCACAGC 609 15075-15097 AD-1309379 ACCCUGGAGAACUGCACGGUU 15119-15139 278 AACCGUGCAGUUCUCCAGGGUCC 610 15117-15139 AD-1309406 GUGGGUGACAACCGUGUCGUU 15149-15169 279 AACGACACGGUUGUCACCCACGC 611 15147-15169 AD-1309433 GACCCAAAGCCUGUGGCCAAU 15176-15196 280 AUUGGCCACAGGCUUUGGGUCCA 612 15174-15196 AD-1309459 CUGCGUGAACAAGCACCUGCU 15202-15222 281 AGCAGGUGCUUGUUCACGCAGGU 613 15200-15222 AD-1309482 UCAAAGUGUCGGACCCGAGCU 15225-15245 282 AGCUCGGGUCCGACACUUUGAUG 614 15223-15245 AD-1309503 CUGUGACUUCCACUAUGAGUU 15250-15270 283 AACUCAUAGUGGAAGUCACAGGG 615 15248-15270 AD-1309526 AGUGCAUCUGCAGCAUGUGGU 15273-15293 284 ACCACAUGCUGCAGAUGCACUCG 616 15271-15293 AD-1309535 CCCACUAUUCCACCUUUGACU 15300-15320 285 AGUCAAAGGUGGAAUAGUGGGAG 617 15298-15320 AD-1309567 ACCUAUGUCCUCAUGAGAGAU 15350-15370 286 AUCUCUCAUGAGGACAUAGGUGC 618 15348-15370 AD-1309595 CACGCUUUGGGAAUCUCAGCU 15378-15398 287 AGCUGAGAUUCCCAAAGCGUGCA 619 15376-15398 AD-1309621 CUGGACAACCACUACUGCACU 15404-15424 288 AGUGCAGUAGUGGUUGUCCAGGU 620 15402-15424 AD-1309647 CCUCAGCAUCCACUACAAGUU 15463-15483 289 AACUUGUAGUGGAUGCUGAGGGC 621 15461-15483 AD-1309678 GUCCUCACUGUCACCAUGGUU 15494-15514 290 AACCAUGGUGACAGUGAGGACGA 622 15492-15514 AD-1309720 AUCCUGUUUGACCAAAUUCCU 15536-15556 291 AGGAAUUUGGUCAAACAGGAUCA 623 15534-15556 AD-1309747 AGCGGUUUCAGCAAGAACGGU 15563-15583 292 ACCGUUCUUGCUGAAACCGCUGC 624 15561-15583 AD-1309780 AUGCGUGUGGACAUUCCUGCU 15614-15634 293 AGCAGGAAUGUCCACACGCAUGG 625 15612-15634 AD-1309804 GUGAGCGUCACCUUCAAUGGU 15641-15661 294 ACCAUUGAAGGUGACGCUCACGC 626 15639-15661 AD-1309844 AGCCUCUUCCACAACAACACU 15689-15709 295 AGUGUUGUUGUGGAAGAGGCUGU 627 15687-15709 AD-1309872 CACCUGCACCAACAACCAGAU 15724-15744 296 AUCUGGUUGUUGGUGCAGGUGCC 628 15722-15744 AD-1309900 UGUCUCCAGCGGGACGGAACU 15752-15772 297 AGUUCCGUCCCGCUGGAGACAGU 629 15750-15772 AD-1309926 CGCCAGUUGCAAGGACAUGGU 15778-15798 298 ACCAUGUCCUUGCAACUGGCGGC 630 15776-15798 AD-1309945 CGACAGCAGAAAGGAUGGCUU 15817-15837 299 AAGCCAUCCUUUCUGCUGUCGGG 631 15815-15837 AD-1309969 CCGCUCUGUGAUCUGAUGCUU 15923-15943 300 AAGCAUCAGAUCACAGAGCGGCU 632 15921-15943 AD-1309993 CAGGUCUUUGCUGAGUGCCAU 15947-15967 301 AUGGCACUCAGCAAAGACCUGGC 633 15945-15967 AD-1310022 CGCCUGCAUCAGCGACCACUU 15997-16017 302 AAGUGGUCGCUGAUGCAGGCGUU 634 15995-16017 AD-1310059 GAGGCUUACGCAGAGCUCUGU 16052-16072 303 ACAGAGCUCUGCGUAAGCCUCCA 635 16050-16072 AD-1310068 AGUGUGCAGUGACUGGCGAGU 16084-16104 304 ACUCGCCAGUCACUGCACACUCC 636 16082-16104 AD-1310124 CACCAAAGUGUACAAGCCAUU 16141-16161 305 AAUGGCUUGUACACUUUGGUGGG 637 16139-16161 AD-1310146 CUGCAACUCUAGGAACCAGAU 16183-16203 306 AUCUGGUUCCUAGAGUUGCAGGU 638 16181-16203 AD-1310192 GACCAGAUCCUCUUCAACGCU 16247-16267 307 AGCGUUGAAGAGGAUCUGGUCCU 639 16245-16267 AD-1310215 CAUGGGCAUCUGCGUGCAGGU 16270-16290 308 ACCUGCACGCAGAUGCCCAUGUG 640 16268-16290 AD-1310234 CGAUGGGUUUCCUAAAUUUCU 16309-16329 309 AGAAAUUUAGGAAACCCAUCGGG 641 16307-16329 AD-1310249 GGUCAGCAACUGCCAGUCCUU 16342-16362 310 AAGGACUGGCAGUUGCUGACCCA 642 16340-16362 AD-1310280 GAGGGUUCAGUGUCGGUGCAU 16373-16393 311 AUGCACCGACACUGAACCCUCGU 643 16371-16393 AD-1310310 CCGGCUUCGUAACCGUGACCU 16446-16466 312 AGGUCACGGUUACGAAGCCGGGA 644 16444-16466 AD-1310328 CGUGUGCAACACAACCACCUU 16507-16527 313 AAGGUGGUUGUGUUGCACACGCA 645 16505-16527 AD-1310345 GGCAGGAGUCCAUCUGCACCU 16557-16577 314 AGGUGCAGAUGGACUCCUGCCCU 646 16555-16577 AD-1310378 CUGCUGUCCCACCUUCCGCUU 16591-16611 315 AAGCGGAAGGUGGGACAGCAGUC 647 16589-16611 AD-1310405 UCAGCUGUGUUCGUACAAUGU 16618-16638 316 ACAUUGUACGAACACAGCUGAGG 648 16616-16638 AD-1310422 UUGGUGCAACCUUCCCAGGCU 16653-16673 317 AGCCUGGGAAGGUUGCACCAACC 649 16651-16673 AD-1310443 UCCCUGCCACAUGUGUACCUU 16678-16698 318 AAGGUACACAUGUGGCAGGGAAG 650 16676-16698 AD-1310466 ACCCAACGGUGCAAUGUCAGU 16719-16739 319 ACUGACAUUGCACCGUUGGGUCC 651 16717-16739 AD-1310493 CCUGCAACAAUACUACCUGUU 16746-16766 320 AACAGGUAGUAUUGUUGCAGGCA 652 16744-16766 AD-1310497 AGGGCUUUGAGUACAAGAGAU 16770-16790 321 AUCUCUUGUACUCAAAGCCCUGG 653 16768-16790 AD-1310552 AGUCCAGCUGAAUGAAACCUU 16852-16872 322 AAGGUUUCAUUCAGCUGGACUGG 654 16850-16872 AD-1310576 CAACAGCCAUGUGGACAACUU 16876-16896 323 AAGUUGUCCACAUGGCUGUUGAC 655 16874-16896 AD-1310599 CCGUGUACCUCUGUGAGGCUU 16899-16919 324 AAGCCUCACAGAGGUACACGGUG 656 16897-16919 AD-1310622 GGUGGAGUCCAUUUGCUGACU 16922-16942 325 AGUCAGCAAAUGGACUCCACCCU 657 16920-16942 AD-1310637 CUGCCCAGAUGUGUCCAGCUU 16957-16977 326 AAGCUGGACACAUCUGGGCAGGA 658 16955-16977 AD-1310665 GCUGCUACUCCUGUGAGGAGU 17004-17024 327 ACUCCUCACAGGAGUAGCAGCAG 659 17002-17024 AD-1310689 CCUGUCAAGUCCGCAUCAACU 17028-17048 328 AGUUGAUGCGGACUUGACAGGAG 660 17026-17048 AD-1310714 CAUCCUGUGGCACCAGGGCUU 17053-17073 329 AAGCCCUGGUGCCACAGGAUGGU 661 17051-17073 AD-1310741 CGAGGUCAACAUCACCUUCUU 17080-17100 330 AAGAAGGUGAUGUUGACCUCGGU 662 17078-17100 AD-1310762 CGUCCAAGUACUCAGCAGAGU 17121-17141 331 ACUCUGCUGAGUACUUGGACGCU 663 17119-17141 AD-1310790 CAUGCAGCACCAGUGCACCUU 17149-17169 332 AAGGUGCACUGGUGCUGCAUGGC 664 17147-17169 AD-1310845 CUUGCACUGUCCUAACGGCUU 17206-17226 333 AAGCCGUUAGGACAGUGCAAGGG 665 17204-17226 AD-1310873 CUGCACACCUACACCCACGUU 17234-17254 334 AACGUGGGUGUAGGUGUGCAGGA 666 17232-17254 AD-1310907 GCACGCCCUUCUGUGUCCCUU 17268-17288 335 AAGGGACACAGAAGGGCGUGCAG 667 17266-17288 AD-1310938 ACUGCUGUCUGAGAACGUUCU 17336-17356 336 AGAACGUUCUCAGACAGCAGUGG 668 17334-17356 AD-1310950 CAUGCUCUGUCCACCUGGAGU 17368-17388 337 ACUCCAGGUGGACAGAGCAUGGG 669 17366-17388 AD-1310979 GCAUUGUCUGAUCAUGAAAAU 17397-17417 338 AUUUUCAUGAUCAGACAAUGCAC 670 17395-17417 AD-1311016 GGCGCCACUCAGGAGUCCUAU 17544-17564 339 AUAGGACUCCUGAGUGGCGCCCU 671 17542-17564 AD-1311052 CUCCCUGAUGUCACUGGGACU 17600-17620 340 AGUCCCAGUGACAUCAGGGAGGG 672 17598-17620 AD-1311075 CUGGAACAAACUAAGCAUGUU 17623-17643 341 AACAUGCUUAGUUUGUUCCAGGG 673 17621-17643 AD-1311115 CACGGAUUCCAGCUGGCCACU 17685-17705 342 AGUGGCCAGCUGGAAUCCGUGCU 674 17683-17705 AD-1311126 GACAGGCUGGUCCAGGCAAGU 17722-17742 343 ACUUGCCUGGACCAGCCUGUCUG 675 17720-17742 AD-1311154 CUGCCAGGAAGCUGCGACAGU 17750-17770 344 ACUGUCGCAGCUUCCUGGCAGCA 676 17748-17770 AD-1311185 CUGCUGCAGGGUAACUCAGGU 17793-17813 345 ACCUGAGUUACCCUGCAGCAGGC 677 17791-17813 AD-1311214 GCAACGGCCAGGUCAGAGAGU 17822-17842 346 ACUCUCUGACCUGGCCGUUGCGA 678 17820-17842 AD-1311231 CCCAGUUUUGCAAAUAAACCU 17878-17898 347 AGGUUUAUUUGCAAAACUGGGCU 679 17876-17898

TABLE 3 Modified Sense and Antisense Strand MUC5B dsRNA Sequences SEQ SEQ ID ID Duplex Name Sense Sequence 5′-3′ NO: Antisense Sequence 5′-3′ NO: AD-1302944 ususggcuCfuGfGfCfggccaugcuuL96 680 asAfsgcaUfgGfCfcgccAfgAfgccaascsa 1011 AD-1302994 cscsgagcUfgGfGfAfgaaugcagguL96 681 asCfscugCfaUfUfcuccCfaGfcucggscsu 1012 AD-1303025 gscsgcguGfaGfCfUfuuguuccacuL96 682 asGfsuggAfaCfAfaagcUfcAfcgcgcscsg 1013 AD-1303054 usgsggcgGfgUfGfUfgcagcaccuuL96 683 asAfsgguGfcUfGfcacaCfcCfgcccasusu 1014 AD-1303066 cscsacuaCfaAfGfAfccuucgacguL96 684 asCfsgucGfaAfGfgucuUfgUfaguggsasa 1015 AD-1303108 cscsuuugCfaAfCfUfacguguucuuL96 685 asAfsgaaCfaCfGfuaguUfgCfaaaggscsc 1016 AD-1303139 gsasggacUfuCfAfAfcguccagcuuL96 686 asAfsgcuGfgAfCfguugAfaGfuccucsgsu 1017 AD-1303190 uscsacccGfuGfUfUfgucaucaaguL96 687 asCfsuugAfuGfAfcaacAfcGfggugascsc 1018 AD-1303222 gsgscuccGfuCfCfUfcaucaaugguL96 688 asCfscauUfgAfUfgaggAfcGfgagccsgsu 1019 AD-1303251 gscsugccUfuAfCfAfgccgcacuguL96 689 asCfsaguGfcGfGfcuguAfaGfgcagcsusc 1020 AD-1303275 gsascuacAfuCfAfAfggucagcauuL96 690 asAfsugcUfgAfCfcuugAfuGfuagucscsc 1021 AD-1303304 gscsugacAfuUfCfCfuguggaacguL96 691 asCfsguuCfcAfCfaggaAfuGfucagcsasc 1022 AD-1303344 gsasgcugGfaUfCfCfcaaauacgcuL96 692 asGfscguAfuUfUfgggaUfcCfagcucscsa 1023 AD-1303368 csasgaccUfgUfGfGfccugugugguL96 693 asCfscacAfcAfGfgccaCfaGfgucugsgsu 1024 AD-1303389 gscscuucAfaCfGfAfguucuaugcuL96 694 asGfscauAfgAfAfcucgUfuGfaaggcscsg 1025 AD-1303422 gsasaccuGfcAfGfAfaguuggauguL96 695 asCfsaucCfaAfCfuucuGfcAfgguucscsc 1026 AD-1303457 usgscacgGfaCfGfAfggagggcauuL96 696 asAfsugcCfcUfCfcucgUfcCfgugcasgsu 1027 AD-1303507 gsusggacAfgCfAfCfugcguaccuuL96 697 asAfsgguAfcGfCfagugCfuGfuccacscsa 1028 AD-1303537 gscscaccUfuUfGfUfggaauacucuL96 698 asGfsaguAfuUfCfcacaAfaGfguggcsasc 1029 AD-1303569 csusggagGfuGfCfCfcugagcucuuL96 699 asAfsgagCfuCfAfgggcAfcCfuccagsusu 1030 AD-1303573 csuscaacAfuGfCfAfgcaccaggauL96 700 asUfsccuGfgUfGfcugcAfuGfuugagsgsg 1031 AD-1303602 ascsccugCfaCfGfGfacaccugcuuL96 701 asAfsgcaGfgUfGfuccgUfgCfagggusgsa 1032 AD-1303626 gsgsaccaCfuGfUfGfuggacggcuuL96 702 asAfsgccGfuCfCfacacAfgUfgguccsusc 1033 AD-1303646 gscsuggaUfgAfCfAfucacgcacuuL96 703 asAfsgugCfgUfGfauguCfaUfccagcsasc 1034 AD-1303679 csasccucCfuUfCfAfacaccaccuuL96 704 asAfsgguGfgUfGfuugaAfgGfaggugscsc 1035 AD-1303705 csusauggCfaGfUfGfccaggaccuuL96 705 asAfsgguCfcUfGfgcacUfgCfcauagscsc 1036 AD-1303746 cscsuaugAfuGfAfGfaaacucuacuL96 706 asGfsuagAfgUfUfucucAfuCfauaggsusg 1037 AD-1303787 usascguuCfuGfUfCfcaagaaauguL96 707 asCfsauuUfcUfUfggacAfgAfacguasgsc 1038 AD-1303811 gsascagcAfgCfUfUfcaccgugcuuL96 708 asAfsgcaCfgGfUfgaagCfuGfcugucsgsg 1039 AD-1303862 asascgagAfaCfUfGfccugaaagcuL96 709 asGfscuuUfcAfGfgcagUfuCfucguusgsu 1040 AD-1303914 uscscucaAfcUfCfCfaucuacacguL96 710 asCfsgugUfaGfAfuggaGfuUfgaggasasc 1041 AD-1303932 gscscaacAfuCfAfCfccuguucacuL96 711 asGfsugaAfcAfGfggugAfuGfuuggcsusg 1042 AD-1303956 uscsgagcUfuCfUfUfcaucgugguuL96 712 asAfsccaCfgAfUfgaagAfaGfcucgasgsg 1043 AD-1303977 csasgcugCfuGfGfUfgcagcugguuL96 713 asAfsccaGfcUfGfcaccAfgCfagcugscsa 1044 AD-1304001 csuscaugCfaGfGfUfguuugucaguL96 714 asCfsugaCfaAfAfcaccUfgCfaugagsusg 1045 AD-1304039 gsusgggaAfcUfUfCfaaccagaacuL96 715 asGfsuucUfgGfUfugaaGfuUfcccacsasc 1046 AD-1304064 usgsacgaCfuUfCfAfcggcccucauL96 716 asUfsgagGfgCfCfgugaAfgUfcgucasgsc 1047 AD-1304077 asgsccuuCfgCfCfAfacaccuggauL96 717 asUfsccaGfgUfGfuuggCfgAfaggcusgsc 1048 AD-1304119 cscsaggaAfcAfGfCfuuugaggacuL96 718 asGfsuccUfcAfAfagcuGfuUfccuggscsa 1049 AD-1304134 gsusggagAfaUfGfAfgaacuacgcuL96 719 asGfscguAfgUfUfcucaUfuCfuccacsasc 1050 AD-1304174 uscsccaaCfaGfUfGfccuucucgcuL96 720 asGfscgaGfaAfGfgcacUfgUfugggasusc 1051 AD-1304202 csusuccaCfuCfGfAfacugcauguuL96 721 asAfscauGfcAfGfuucgAfgUfggaagsgsg 1052 AD-1304226 csasccugCfaAfCfUfgugagcggauL96 722 asUfsccgCfuCfAfcaguUfgCfaggugsusc 1053 AD-1304262 cscsuccuAfuGfUfGfcacgccuguuL96 723 asAfscagGfcGfUfgcacAfuAfggaggsasc 1054 AD-1304286 gsgsgcguAfcAfGfCfucagcgacuuL96 724 asAfsgucGfcUfGfagcuGfuAfcgcccsusu 1055 AD-1304322 ascscaagUfaCfAfUfgcagaacuguL96 725 asCfsaguUfcUfGfcaugUfaCfuuggusgsc 1056 AD-1304326 asasguccCfaGfCfGfcuacgccuauL96 726 asUfsaggCfgUfAfgcgcUfgGfgacuusgsg 1057 AD-1304352 gsgsaugcCfuGfCfCfagcccacuuuL96 727 asAfsaguGfgGfCfuggcAfgGfcauccsasc 1058 AD-1304386 uscsaccuGfcAfGfCfguuuccuucuL96 728 asGfsaagGfaAfAfcgcuGfcAfggugascsg 1059 AD-1304415 gsgsgcacCfuUfCfCfucaaugacguL96 729 asCfsgucAfuUfGfaggaAfgGfugcccsgsc 1060 AD-1304454 csusggcuCfcUfGfGfagaggugguuL96 730 asAfsccaCfcUfCfuccaGfgAfgccagscsa 1061 AD-1304482 cscsguguGfuUfCfAfuguacggguuL96 731 asAfscccGfuAfCfaugaAfcAfcacggscsg 1062 AD-1304524 cscsucucUfgCfAfGfaaaagcacauL96 732 asUfsgugCfuUfUfucugCfaGfagaggscsu 1063 AD-1304546 cscsuggaCfuGfCfAfgcaacagcuuL96 733 asAfsgcuGfuUfGfcugcAfgUfccaggsusa 1064 AD-1304596 gscsuguuUfcAfGfCfacacacugcuL96 734 asGfscagUfgUfGfugcuGfaAfacagcscsc 1065 AD-1304624 csusgcauUfgCfCfGfaggaggacuuL96 735 asAfsgucCfuCfCfucggCfaAfugcagscsc 1066 AD-1304646 csasccuaCfaAfGfCfcuggagagauL96 736 asUfscucUfcCfAfggcuUfgUfaggugsgsc 1067 AD-1304676 csgsacugCfaAfCfAfccugcaccuuL96 737 asAfsgguGfcAfGfguguUfgCfagucgsasc 1068 AD-1304700 gsasaccgGfaGfGfUfgggagugcauL96 738 asUfsgcaCfuCfCfcaccUfcCfgguucscsu 1069 AD-1304732 usgsgccaCfuUfCfAfucaccuuuguL96 739 asCfsaaaGfgUfGfaugaAfgUfggccasusc 1070 AD-1304756 csgsaucgCfuAfCfAfgcuuugaaguL96 740 asCfsuucAfaAfGfcuguAfgCfgaucgscsc 1071 AD-1304779 gscsugcgAfgUfAfCfaucuuggccuL96 741 asGfsgccAfaGfAfuguaCfuCfgcagcsusg 1072 AD-1304818 uscscgcaUfcGfUfCfaccgagaacuL96 742 asGfsuucUfcGfGfugacGfaUfgcggasasg 1073 AD-1304850 asasggccAfuCfAfAfgcucuucguuL96 743 asAfscgaAfgAfGfcuugAfuGfgccuusgsg 1074 AD-1304873 gsasgcuaCfgAfGfCfugauccuccuL96 744 asGfsgagGfaUfCfagcuCfgUfagcucsusc 1075 AD-1304882 gsasccuuUfaAfGfGfcgguggcgauL96 745 asUfscgcCfaCfCfgccuUfaAfaggucscsc 1076 AD-1304894 csascccuAfcAfAfGfauacgcuacuL96 746 asGfsuagCfgUfAfucuuGfuAfgggugsgsg 1077 AD-1304902 asuscuucCfuGfGfUfcaucgagacuL96 747 asGfsucuCfgAfUfgaccAfgGfaagauscsc 1078 AD-1304953 csasgcguGfuUfCfAfuccgacugcuL96 748 asGfscagUfcGfGfaugaAfcAfcgcugsgsu 1079 AD-1304977 gsgsacuaCfaAfGfGfgcagggucuuL96 749 asAfsgacCfcUfGfcccuUfgUfaguccsusg 1080 AD-1305014 gsascaauGfcCfAfUfcaaugacuuuL96 750 asAfsaguCfaUfUfgaugGfcAfuugucsgsu 1081 AD-1305043 gscsacugGfaGfUfUfugggaacaguL96 751 asCfsuguUfcCfCfaaacUfcCfagugcsgsu 1082 AD-1305082 csasgaagCfaGfUfGfcagcauccuuL96 752 asAfsggaUfgCfUfgcacUfgCfuucugsgsg 1083 AD-1305104 cscsagguUfgAfCfUfccaccaaguuL96 753 asAfscuuGfgUfGfgaguCfaAfccuggsgsa 1084 AD-1305128 csgsaggcCfuGfCfGfugaacgacguL96 754 asCfsgucGfuUfCfacgcAfgGfccucgsusa 1085 AD-1305170 ascsugcgAfgUfGfUfuucugcacguL96 755 asCfsgugCfaGfAfaacaCfuCfgcaguscsg 1086 AD-1305212 usgsugugUfgUfCfCfuggcggacuuL96 756 asAfsgucCfgCfCfaggaCfaCfacacasgsg 1087 AD-1305228 usgsuucuGfuGfAfCfuucuacaacuL96 757 asGfsuugUfaGfAfagucAfcAfgaacasasg 1088 AD-1305240 csusgugaGfuGfGfCfacuaccagcuL96 758 asGfscugGfuAfGfugccAfcUfcacagscsc 1089 AD-1305297 gscsugcuAfcCfCfGfaagugcccauL96 759 asUfsgggCfaCfUfucggGfuAfgcagescsu 1090 AD-1305321 asgscccuUfcUfUfCfaaugaggacuL96 760 asGfsuccUfcAfUfugaaGfaAfgggcusgsg 1091 AD-1305347 asasgugcGfuGfGfCfccagugugguL96 761 asCfscacAfcUfGfggccAfcGfcacuuscsa 1092 AD-1305370 csusacgaCfaAfGfGfacggaaacuuL96 762 asAfsguuUfcCfGfuccuUfgUfcguagscsa 1093 AD-1305393 asusgacgUfcGfGfUfgcaagggucuL96 763 asGfsaccCfuUfGfcaccGfaCfgucausasg 1094 AD-1305410 cscsagagCfuGfUfAfacugcacacuL96 764 asGfsuguGfcAfGfuuacAfgCfucuggscsa 1095 AD-1305441 csasgugcGfcUfCfAfcagccuugauL96 765 asUfscaaGfgCfUfgugaGfcGfcacugsgsa 1096 AD-1305471 usgscaccUfaUfGfAfggacaggacuL96 766 asGfsuccUfgUfCfcucaUfaGfgugcasgsg 1097 AD-1305501 csasggacGfuCfAfUfcuacaacacuL96 767 asGfsuguUfgUfAfgaugAfcGfuccugsgsu 1098 AD-1305536 csgsccugCfuUfGfAfucgccaucuuL96 768 asAfsgauGfgCfGfaucaAfgCfaggcgscsc 1099 AD-1305570 ascscaucAfuCfAfGfgaaggcuguuL96 769 asAfscagCfcUfUfccugAfuGfauggusgsc 1100 AD-1305610 cscsacaaCfgCfCfAfuucaccuucuL96 770 asGfsaagGfuGfAfauggCfgUfuguggscsu 1101 AD-1305643 uscscaccGfuGfUfGfuguccgcgauL96 771 asUfscgcGfgAfCfacacAfcGfguggasgsa 1102 AD-1305672 gsusccagCfuGfGfUfacaaugggcuL96 772 asGfscccAfuUfGfuaccAfgCfuggacscsa 1103 AD-1305701 gsascuuuGfaGfAfCfguuugaaaauL96 773 asUfsuuuCfaAfAfcgucUfcAfaagucsusc 1104 AD-1305731 asgsagggUfaCfCfAfgguaugcccuL96 774 asGfsggcAfuAfCfcuggUfaCfccucuscsu 1105 AD-1305754 gscsuggcUfgAfCfAfucgagugccuL96 775 asGfsgcaCfuCfGfauguCfaGfccagcsasc 1106 AD-1305771 csusucccGfaCfAfUfgccgcuggauL96 776 asUfsccaGfcGfGfcaugUfcGfggaagscsu 1107 AD-1305801 csasggugGfaCfUfGfugaccgcauuL96 777 asAfsugcGfgUfCfacagUfcCfaccugscsu 1108 AD-1305817 csgsccaaCfaGfCfCfaacagagucuL96 778 asGfsacuCfuGfUfuggcUfgUfuggcgscsa 1109 AD-1305821 csuscuguCfaCfGfAfcuacgagcuuL96 779 asAfsgcuCfgUfAfgucgUfgAfcagagscsg 1110 AD-1305847 uscsucugCfuGfCfGfaauacgugcuL96 780 asGfscacGfuAfUfucgcAfgCfagagasasc 1111 AD-1305889 csascggaGfcCfUfGfcugugccuauL96 781 asUfsaggCfaCfAfgcagGfcUfccgugscsu 1112 AD-1305898 asgsaccaCfaGfCfAfaccgaaaaguL96 782 asCfsuuuUfcGfGfuugcUfgUfggucusgsg 1113 AD-1305930 csuscgcaGfaCfUfGfgguccagcuuL96 783 asAfsgcuGfgAfCfccagUfcUfgcgagsgsu 1114 AD-1305948 gsusggacAfgAfGfUfgguuugauguL96 784 asCfsaucAfaAfCfcacuCfuGfuccacsusg 1115 AD-1305958 csasagucUfgAfAfCfaacuuggaguL96 785 asCfsuccAfaGfUfuguuCfaGfacuugsgsg 1116 AD-1305966 ususgaguCfcUfAfCfgauaagaucuL96 786 asGfsaucUfuAfUfcguaGfgAfcucaascsg 1117 AD-1306008 gscsagccUfaAfGfGfacauagaguuL96 787 asAfscucUfaUfGfuccuUfaGfgcugcsusg 1118 AD-1306030 csusggacCfcUfGfGfcacaggugguL96 788 asCfscacCfuGfUfgccaGfgGfuccagsusu 1119 AD-1306042 gscsacugUfgAfCfGfuccacuucguL96 789 asCfsgaaGfuGfGfacguCfaCfagugcsasc 1120 AD-1306069 gsusgcagGfaAfCfUfgggagcagguL96 790 asCfscugCfuCfCfcaguUfcCfugcacsasc 1121 AD-1306099 csasagauGfuGfCfUfacaacuacauL96 791 asUfsguaGfuUfGfuagcAfcAfucuugsasa 1122 AD-1306132 csusgcugCfaGfUfGfacgaccacuuL96 792 asAfsgugGfuCfGfucacUfgCfagcagsasg 1123 AD-1306144 csgsaccaCfaGfAfGfcuggagacguL96 793 asCfsgucUfcCfAfgcucUfgUfggucgsgsu 1124 AD-1306168 ascsccagGfcCfCfUfguucucaacuL96 794 asGfsuugAfgAfAfcaggGfcCfugggusgsg 1125 AD-1306198 csuscucaGfaAfGfGfacugacaucuL96 795 asGfsaugUfcAfGfuccuUfcUfgagagsgsg 1126 AD-1306200 csasgauaCfaCfAfAfgcacccuuguL96 796 asCfsaagGfgUfGfcuugUfgUfaucugsgsg 1127 AD-1306235 gscsuccaCfaGfAfAfcccacugucuL96 797 asGfsacaGfuGfGfguucUfgUfggagcscsu 1128 AD-1306254 csascccuUfcCfAfAfcacgcucaguL96 798 asCfsugaGfcGfUfguugGfaAfgggugsgsa 1129 AD-1306278 csasacaaCfaAfUfGfgcaaccuccuL96 799 asGfsgagGfuUfGfccauUfgUfuguugsgsg 1130 AD-1306324 ascscgcuUfcCfAfAfagagccgcuuL96 800 asAfsgcgGfcUfCfuuugGfaAfgcggusgsc 1131 AD-1306359 cscsaacaCfuCfAfCfgagcgagcuuL96 801 asAfsgcuCfgCfUfcgugAfgUfguuggscsg 1132 AD-1306382 csasccucUfcAfGfGfccgagaccauL96 802 asUfsgguCfuCfGfgccuGfaGfaggugsgsa 1133 AD-1306406 cscsaggaCfaGfAfGfacgacaauguL96 803 asCfsauuGfuCfGfucucUfgUfccuggsgsc 1134 AD-1306411 csusugacUfaAfCfAfccaccaccauL96 804 asUfsgguGfgUfGfguguUfaGfucaagsgsg 1135 AD-1306442 csusgucaAfcCfGfAfagugugaguuL96 805 asAfscucAfcAfCfuucgGfuUfgacagscsg 1136 AD-1306478 csgsuggaCfuUfCfCfcaaccucaguL96 806 asCfsugaGfgUfUfgggaAfgUfccacgsusc 1137 AD-1306483 ascsauggAfaAfCfUfuuugaaaacuL96 807 asGfsuuuUfcAfAfaaguUfuCfcauguscsc 1138 AD-1306514 gscsaccaAfaGfAfGfcauagaguguL96 808 asCfsacuCfuAfUfgcucUfuUfggugcscsc 1139 AD-1306535 gsgsuaagCfaUfCfGfaccaggucguL96 809 asCfsgacCfuGfGfucgaUfgCfuuaccsusc 1140 AD-1306563 csusgaccUfgCfAfGfccuggagacuL96 810 asGfsucuCfcAfGfgcugCfaGfgucagscsa 1141 AD-1306575 usgscaagAfaCfGfAfagaccagacuL96 811 asGfsucuGfgUfCfuucgUfuCfuugcasgsg 1142 AD-1306603 uscsaacaUfgUfGfCfuucaacuacuL96 812 asGfsuagUfuGfAfagcaCfaUfguugasasc 1143 AD-1306627 usgscgugUfgCfUfUfugcugugacuL96 813 asGfsucaCfaGfCfaaagCfaCfacgcascsg 1144 AD-1306673 csusccacCfcUfGfAfgaacagcucuL96 814 asGfsagcUfgUfUfcucaGfgGfuggagsgsu 1145 AD-1306676 uscsccaaAfgUfGfCfugaccaccauL96 815 asUfsgguGfgUfCfagcaCfuUfugggasgsg 1146 AD-1306698 ususccucCfcUfGfGfgcaccaccuuL96 816 asAfsgguGfgUfGfcccaGfgGfaggaasgsa 1147 AD-1306717 usgsccaaCfuAfCfCfacaaccacguL96 817 asCfsgugGfuUfGfugguAfgUfuggcascsu 1148 AD-1307191 csascaccCfaCfAfAfccagaggcuuL96 818 asAfsgccUfcUfGfguugUfgGfgugugsgsu 1149 AD-1307354 gsasccugGfaUfCfCfucacaaagcuL96 819 asGfscuuUfgUfGfaggaUfcCfaggucsgsu 1150 AD-1308029 csusaccaGfcGfUfUfacacccaucuL96 820 asGfsaugGfgUfGfuaacGfcUfgguagscsu 1151 AD-1308183 cscsacaaCfaGfCfCfacuacgacuuL96 821 asAfsgucGfuAfGfuggcUfgUfuguggsusc 1152 AD-1308294 csasgcucCfaAfAfGfccacucccuuL96 822 asAfsgggAfgUfGfgcuuUfgGfagcugsgsu 1153 AD-1334092 gscsccuuCfcAfGfCfacugagaaguL96 823 asCfsuucUfcAfGfugcuGfgAfagggcsgsg 1154 AD-1308403 cscsuaucAfcAfGfAfccaccacacuL96 824 asGfsuguGfgUfGfgucuGfuGfauaggscsg 1155 AD-1308420 gsgsccacCfaUfGfUfccacagccauL96 825 asUfsggcUfgUfGfgacaUfgGfuggccsgsu 1156 AD-1308440 cscsuccuCfcAfCfUfccagagacuuL96 826 asAfsgucUfcUfGfgaguGfgAfggaggsgsu 1157 AD-1334093 csascaccUfcCfAfCfagugcuuacuL96 827 asGfsuaaGfcAfCfugugGfaGfgugugsgsg 1158 AD-1308488 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asGfsagaAfaAfAfcuguCfcAfaaugcscsc 1268 AD-1309259 gsasagucAfuCfUfAfcaauaagacuL96 937 asGfsucuUfaUfUfguagAfuGfacuucscsc 1269 AD-1309287 csusgccaUfuUfCfUfacgcaguguuL96 938 asAfscacUfgCfGfuagaAfaUfggcagscsc 1270 AD-1309315 csascuguGfaCfAfUfugaccgcuuuL96 939 asAfsagcGfgUfCfaaugUfcAfcagugscsu 1271 AD-1309337 usgsugacAfaUfGfCfcaucccucuuL96 940 asAfsgagGfgAfUfggcaUfuGfucacasgsc 1272 AD-1309379 ascsccugGfaGfAfAfcugcacgguuL96 941 asAfsccgUfgCfAfguucUfcCfaggguscsc 1273 AD-1309406 gsusggguGfaCfAfAfccgugucguuL96 942 asAfscgaCfaCfGfguugUfcAfcccacsgsc 1274 AD-1309433 gsascccaAfaGfCfCfuguggccaauL96 943 asUfsuggCfcAfCfaggcUfuUfgggucscsa 1275 AD-1309459 csusgcguGfaAfCfAfagcaccugcuL96 944 asGfscagGfuGfCfuuguUfcAfcgcagsgsu 1276 AD-1309482 uscsaaagUfgUfCfGfgacccgagcuL96 945 asGfscucGfgGfUfccgaCfaCfuuugasusg 1277 AD-1309503 csusgugaCfuUfCfCfacuaugaguuL96 946 asAfscucAfuAfGfuggaAfgUfcacagsgsg 1278 AD-1309526 asgsugcaUfcUfGfCfagcaugugguL96 947 asCfscacAfuGfCfugcaGfaUfgcacuscsg 1279 AD-1309535 cscscacuAfuUfCfCfaccuuugacuL96 948 asGfsucaAfaGfGfuggaAfuAfgugggsasg 1280 AD-1309567 ascscuauGfuCfCfUfcaugagagauL96 949 asUfscucUfcAfUfgaggAfcAfuaggusgsc 1281 AD-1309595 csascgcuUfuGfGfGfaaucucagcuL96 950 asGfscugAfgAfUfucccAfaAfgcgugscsa 1282 AD-1309621 csusggacAfaCfCfAfcuacugcacuL96 951 asGfsugcAfgUfAfguggUfuGfuccagsgsu 1283 AD-1309647 cscsucagCfaUfCfCfacuacaaguuL96 952 asAfscuuGfuAfGfuggaUfgCfugaggsgsc 1284 AD-1309678 gsusccucAfcUfGfUfcaccaugguuL96 953 asAfsccaUfgGfUfgacaGfuGfaggacsgsa 1285 AD-1309720 asusccugUfuUfGfAfccaaauuccuL96 954 asGfsgaaUfuUfGfgucaAfaCfaggauscsa 1286 AD-1309747 asgscgguUfuCfAfGfcaagaacgguL96 955 asCfscguUfcUfUfgcugAfaAfccgcusgsc 1287 AD-1309780 asusgcguGfuGfGfAfcauuccugcuL96 956 asGfscagGfaAfUfguccAfcAfcgcausgsg 1288 AD-1309804 gsusgagcGfuCfAfCfcuucaaugguL96 957 asCfscauUfgAfAfggugAfcGfcucacsgsc 1289 AD-1309844 asgsccucUfuCfCfAfcaacaacacuL96 958 asGfsuguUfgUfUfguggAfaGfaggcusgsu 1290 AD-1309872 csasccugCfaCfCfAfacaaccagauL96 959 asUfscugGfuUfGfuuggUfgCfaggugscsc 1291 AD-1309900 usgsucucCfaGfCfGfggacggaacuL96 960 asGfsuucCfgUfCfccgcUfgGfagacasgsu 1292 AD-1309926 csgsccagUfuGfCfAfaggacaugguL96 961 asCfscauGfuCfCfuugcAfaCfuggcgsgsc 1293 AD-1309945 csgsacagCfaGfAfAfaggauggcuuL96 962 asAfsgccAfuCfCfuuucUfgCfugucgsgsg 1294 AD-1309969 cscsgcucUfgUfGfAfucugaugcuuL96 963 asAfsgcaUfcAfGfaucaCfaGfagcggscsu 1295 AD-1309993 csasggucUfuUfGfCfugagugccauL96 964 asUfsggcAfcUfCfagcaAfaGfaccugsgsc 1296 AD-1310022 csgsccugCfaUfCfAfgcgaccacuuL96 965 asAfsgugGfuCfGfcugaUfgCfaggcgsusu 1297 AD-1310059 gsasggcuUfaCfGfCfagagcucuguL96 966 asCfsagaGfcUfCfugcgUfaAfgccucscsa 1298 AD-1310068 asgsugugCfaGfUfGfacuggcgaguL96 967 asCfsucgCfcAfGfucacUfgCfacacuscsc 1299 AD-1310124 csasccaaAfgUfGfUfacaagccauuL96 968 asAfsuggCfuUfGfuacaCfuUfuggugsgsg 1300 AD-1310146 csusgcaaCfuCfUfAfggaaccagauL96 969 asUfscugGfuUfCfcuagAfgUfugcagsgsu 1301 AD-1310192 gsasccagAfuCfCfUfcuucaacgcuL96 970 asGfscguUfgAfAfgaggAfuCfuggucscsu 1302 AD-1310215 csasugggCfaUfCfUfgcgugcagguL96 971 asCfscugCfaCfGfcagaUfgCfccaugsusg 1303 AD-1310234 csgsauggGfuUfUfCfcuaaauuucuL96 972 asGfsaaaUfuUfAfggaaAfcCfcaucgsgsg 1304 AD-1310249 gsgsucagCfaAfCfUfgccaguccuuL96 973 asAfsggaCfuGfGfcaguUfgCfugaccscsa 1305 AD-1310280 gsasggguUfcAfGfUfgucggugcauL96 974 asUfsgcaCfcGfAfcacuGfaAfcccucsgsu 1306 AD-1310310 cscsggcuUfcGfUfAfaccgugaccuL96 975 asGfsgucAfcGfGfuuacGfaAfgccggsgsa 1307 AD-1310328 csgsugugCfaAfCfAfcaaccaccuuL96 976 asAfsgguGfgUfUfguguUfgCfacacgscsa 1308 AD-1310345 gsgscaggAfgUfCfCfaucugcaccuL96 977 asGfsgugCfaGfAfuggaCfuCfcugccscsu 1309 AD-1310378 csusgcugUfcCfCfAfccuuccgcuuL96 978 asAfsgcgGfaAfGfguggGfaCfagcagsusc 1310 AD-1310405 uscsagcuGfuGfUfUfcguacaauguL96 979 asCfsauuGfuAfCfgaacAfcAfgcugasgsg 1311 AD-1310422 ususggugCfaAfCfCfuucccaggcuL96 980 asGfsccuGfgGfAfagguUfgCfaccaascsc 1312 AD-1310443 uscsccugCfcAfCfAfuguguaccuuL96 981 asAfsgguAfcAfCfauguGfgCfagggasasg 1313 AD-1310466 ascsccaaCfgGfUfGfcaaugucaguL96 982 asCfsugaCfaUfUfgcacCfgUfuggguscsc 1314 AD-1310493 cscsugcaAfcAfAfUfacuaccuguuL96 983 asAfscagGfuAfGfuauuGfuUfgcaggscsa 1315 AD-1310497 asgsggcuUfuGfAfGfuacaagagauL96 984 asUfscucUfuGfUfacucAfaAfgcccusgsg 1316 AD-1310552 asgsuccaGfcUfGfAfaugaaaccuuL96 985 asAfsgguUfuCfAfuucaGfcUfggacusgsg 1317 AD-1310576 csasacagCfcAfUfGfuggacaacuuL96 986 asAfsguuGfuCfCfacauGfgCfuguugsasc 1318 AD-1310599 cscsguguAfcCfUfCfugugaggcuuL96 987 asAfsgccUfcAfCfagagGfuAfcacggsusg 1319 AD-1310622 gsgsuggaGfuCfCfAfuuugcugacuL96 988 asGfsucaGfcAfAfauggAfcUfccaccscsu 1320 AD-1310637 csusgcccAfgAfUfGfuguccagcuuL96 989 asAfsgcuGfgAfCfacauCfuGfggcagsgsa 1321 AD-1310665 gscsugcuAfcUfCfCfugugaggaguL96 990 asCfsuccUfcAfCfaggaGfuAfgcagcsasg 1322 AD-1310689 cscsugucAfaGfUfCfcgcaucaacuL96 991 asGfsuugAfuGfCfggacUfuGfacaggsasg 1323 AD-1310714 csasuccuGfuGfGfCfaccagggcuuL96 992 asAfsgccCfuGfGfugccAfcAfggaugsgsu 1324 AD-1310741 csgsagguCfaAfCfAfucaccuucuuL96 993 asAfsgaaGfgUfGfauguUfgAfccucgsgsu 1325 AD-1310762 csgsuccaAfgUfAfCfucagcagaguL96 994 asCfsucuGfcUfGfaguaCfuUfggacgscsu 1326 AD-1310790 csasugcaGfcAfCfCfagugcaccuuL96 995 asAfsgguGfcAfCfugguGfcUfgcaugsgsc 1327 AD-1310845 csusugcaCfuGfUfCfcuaacggcuuL96 996 asAfsgccGfuUfAfggacAfgUfgcaagsgsg 1328 AD-1310873 csusgcacAfcCfUfAfcacccacguuL96 997 asAfscguGfgGfUfguagGfuGfugcagsgsa 1329 AD-1310907 gscsacgcCfcUfUfCfugugucccuuL96 998 asAfsgggAfcAfCfagaaGfgGfcgugcsasg 1330 AD-1310938 ascsugcuGfuCfUfGfagaacguucuL96 999 asGfsaacGfuUfCfucagAfcAfgcagusgsg 1331 AD-1310950 csasugcuCfuGfUfCfcaccuggaguL96 1000 asCfsuccAfgGfUfggacAfgAfgcaugsgsg 1332 AD-1310979 gscsauugUfcUfGfAfucaugaaaauL96 1001 asUfsuuuCfaUfGfaucaGfaCfaaugcsasc 1333 AD-1311016 gsgscgccAfcUfCfAfggaguccuauL96 1002 asUfsaggAfcUfCfcugaGfuGfgcgccscsu 1334 AD-1311052 csuscccuGfaUfGfUfcacugggacuL96 1003 asGfsuccCfaGfUfgacaUfcAfgggagsgsg 1335 AD-1311075 csusggaaCfaAfAfCfuaagcauguuL96 1004 asAfscauGfcUfUfaguuUfgUfuccagsgsg 1336 AD-1311115 csascggaUfuCfCfAfgcuggccacuL96 1005 asGfsuggCfcAfGfcuggAfaUfccgugscsu 1337 AD-1311126 gsascaggCfuGfGfUfccaggcaaguL96 1006 asCfsuugCfcUfGfgaccAfgCfcugucsusg 1338 AD-1311154 csusgccaGfgAfAfGfcugcgacaguL96 1007 asCfsuguCfgCfAfgcuuCfcUfggcagscsa 1339 AD-1311185 csusgcugCfaGfGfGfuaacucagguL96 1008 asCfscugAfgUfUfacccUfgCfagcagsgsc 1340 AD-1311214 gscsaacgGfcCfAfGfgucagagaguL96 1009 asCfsucuCfuGfAfccugGfcCfguugcsgsa 1341 AD-1311231 cscscaguUfuUfGfCfaaauaaaccuL96 1010 asGfsguuUfaUfUfugcaAfaAfcugggscsu 1342 SEQ ID Duplex Name mRNA target sequence NO: AD-1302944 UGUUGGCUCUGGCGGCCAUGCUC 1343 AD-1302994 AGCCGAGCUGGGAGAAUGCAGGG 1344 AD-1303025 CGGCGCGUGAGCUUUGUUCCACC 1345 AD-1303054 AAUGGGCGGGUGUGCAGCACCUG 1346 AD-1303066 UUCCACUACAAGACCUUCGACGG 1347 AD-1303108 GGCCUUUGCAACUACGUGUUCUC 1348 AD-1303139 ACGAGGACUUCAACGUCCAGCUA 1349 AD-1303190 GGUCACCCGUGUUGUCAUCAAGG 1350 AD-1303222 ACGGCUCCGUCCUCAUCAAUGGG 1351 AD-1303251 GAGCUGCCUUACAGCCGCACUGG 1352 AD-1303275 GGGACUACAUCAAGGUCAGCAUC 1353 AD-1303304 GUGCUGACAUUCCUGUGGAACGG 1354 AD-1303344 UGGAGCUGGAUCCCAAAUACGCC 1355 AD-1303368 ACCAGACCUGUGGCCUGUGUGGG 1356 AD-1303389 CGGCCUUCAACGAGUUCUAUGCC 1357 AD-1303422 GGGAACCUGCAGAAGUUGGAUGG 1358 AD-1303457 ACUGCACGGACGAGGAGGGCAUC 1359 AD-1303507 UGGUGGACAGCACUGCGUACCUG 1360 AD-1303537 GUGCCACCUUUGUGGAAUACUCA 1361 AD-1303569 AACUGGAGGUGCCCUGAGCUCUG 1362 AD-1303573 CCCUCAACAUGCAGCACCAGGAG 1363 AD-1303602 UCACCCUGCACGGACACCUGCUC 1364 AD-1303626 GAGGACCACUGUGUGGACGGCUG 1365 AD-1303646 GUGCUGGAUGACAUCACGCACUC 1366 AD-1303679 GGCACCUCCUUCAACACCACCUG 1367 AD-1303705 GGCUAUGGCAGUGCCAGGACCUG 1368 AD-1303746 CACCUAUGAUGAGAAACUCUACG 1369 AD-1303787 GCUACGUUCUGUCCAAGAAAUGU 1370 AD-1303811 CCGACAGCAGCUUCACCGUGCUG 1371 AD-1303862 ACAACGAGAACUGCCUGAAAGCG 1372 AD-1303914 GUUCCUCAACUCCAUCUACACGC 1373 AD-1303932 CAGCCAACAUCACCCUGUUCACA 1374 AD-1303956 CCUCGAGCUUCUUCAUCGUGGUG 1375 AD-1303977 UGCAGCUGCUGGUGCAGCUGGUG 1376 AD-1304001 CACUCAUGCAGGUGUUUGUCAGG 1377 AD-1304039 GUGUGGGAACUUCAACCAGAACC 1378 AD-1304064 GCUGACGACUUCACGGCCCUCAG 1379 AD-1304077 GCAGCCUUCGCCAACACCUGGAA 1380 AD-1304119 UGCCAGGAACAGCUUUGAGGACC 1381 AD-1304134 GUGUGGAGAAUGAGAACUACGCC 1382 AD-1304174 GAUCCCAACAGUGCCUUCUCGCG 1383 AD-1304202 CCCUUCCACUCGAACUGCAUGUU 1384 AD-1304226 GACACCUGCAACUGUGAGCGGAG 1385 AD-1304262 GUCCUCCUAUGUGCACGCCUGUG 1386 AD-1304286 AAGGGCGUACAGCUCAGCGACUG 1387 AD-1304322 GCACCAAGUACAUGCAGAACUGC 1388 AD-1304326 CCAAGUCCCAGCGCUACGCCUAC 1389 AD-1304352 GUGGAUGCCUGCCAGCCCACUUG 1390 AD-1304386 CGUCACCUGCAGCGUUUCCUUCG 1391 AD-1304415 GCGGGCACCUUCCUCAAUGACGC 1392 AD-1304454 UGCUGGCUCCUGGAGAGGUGGUG 1393 AD-1304482 CGCCGUGUGUUCAUGUACGGGUG 1394 AD-1304524 AGCCUCUCUGCAGAAAAGCACAG 1395 AD-1304546 UACCUGGACUGCAGCAACAGCUC 1396 AD-1304596 GGGCUGUUUCAGCACACACUGCG 1397 AD-1304624 GGCUGCAUUGCCGAGGAGGACUG 1398 AD-1304646 GCCACCUACAAGCCUGGAGAGAC 1399 AD-1304676 GUCGACUGCAACACCUGCACCUG 1400 AD-1304700 AGGAACCGGAGGUGGGAGUGCAG 1401 AD-1304732 GAUGGCCACUUCAUCACCUUUGA 1402 AD-1304756 GGCGAUCGCUACAGCUUUGAAGG 1403 AD-1304779 CAGCUGCGAGUACAUCUUGGCCC 1404 AD-1304818 CUUCCGCAUCGUCACCGAGAACA 1405 AD-1304850 CCAAGGCCAUCAAGCUCUUCGUG 1406 AD-1304873 GAGAGCUACGAGCUGAUCCUCCA 1407 AD-1304882 GGGACCUUUAAGGCGGUGGCGAG 1408 AD-1304894 CCCACCCUACAAGAUACGCUACA 1409 AD-1304902 GGAUCUUCCUGGUCAUCGAGACC 1410 AD-1304953 ACCAGCGUGUUCAUCCGACUGCA 1411 AD-1304977 CAGGACUACAAGGGCAGGGUCUG 1412 AD-1305014 ACGACAAUGCCAUCAAUGACUUU 1413 AD-1305043 ACGCACUGGAGUUUGGGAACAGC 1414 AD-1305082 CCCAGAAGCAGUGCAGCAUCCUC 1415 AD-1305104 UCCCAGGUUGACUCCACCAAGUA 1416 AD-1305128 UACGAGGCCUGCGUGAACGACGC 1417 AD-1305170 CGACUGCGAGUGUUUCUGCACGG 1418 AD-1305212 CCUGUGUGUGUCCUGGCGGACUC 1419 AD-1305228 CUUGUUCUGUGACUUCUACAACC 1420 AD-1305240 GGCUGUGAGUGGCACUACCAGCC 1421 AD-1305297 AGGCUGCUACCCGAAGUGCCCAC 1422 AD-1305321 CCAGCCCUUCUUCAAUGAGGACC 1423 AD-1305347 UGAAGUGCGUGGCCCAGUGUGGC 1424 AD-1305370 UGCUACGACAAGGACGGAAACUA 1425 AD-1305393 CUAUGACGUCGGUGCAAGGGUCC 1426 AD-1305410 UGCCAGAGCUGUAACUGCACACC 1427 AD-1305441 UCCAGUGCGCUCACAGCCUUGAG 1428 AD-1305471 CCUGCACCUAUGAGGACAGGACC 1429 AD-1305501 ACCAGGACGUCAUCUACAACACC 1430 AD-1305536 GGCGCCUGCUUGAUCGCCAUCUG 1431 AD-1305570 GCACCAUCAUCAGGAAGGCUGUG 1432 AD-1305610 AGCCACAACGCCAUUCACCUUCA 1433 AD-1305643 UCUCCACCGUGUGUGUCCGCGAG 1434 AD-1305672 UGGUCCAGCUGGUACAAUGGGCA 1435 AD-1305701 GAGACUUUGAGACGUUUGAAAAC 1436 AD-1305731 AGAGAGGGUACCAGGUAUGCCCU 1437 AD-1305754 GUGCUGGCUGACAUCGAGUGCCG 1438 AD-1305771 AGCUUCCCGACAUGCCGCUGGAG 1439 AD-1305801 AGCAGGUGGACUGUGACCGCAUG 1440 AD-1305817 UGCGCCAACAGCCAACAGAGUCC 1441 AD-1305821 CGCUCUGUCACGACUACGAGCUG 1442 AD-1305847 GUUCUCUGCUGCGAAUACGUGCC 1443 AD-1305889 AGCACGGAGCCUGCUGUGCCUAC 1444 AD-1305898 CCAGACCACAGCAACCGAAAAGA 1445 AD-1305930 ACCUCGCAGACUGGGUCCAGCUC 1446 AD-1305948 CAGUGGACAGAGUGGUUUGAUGA 1447 AD-1305958 CCCAAGUCUGAACAACUUGGAGG 1448 AD-1305966 CGUUGAGUCCUACGAUAAGAUCA 1449 AD-1306008 CAGCAGCCUAAGGACAUAGAGUG 1450 AD-1306030 AACUGGACCCUGGCACAGGUGGG 1451 AD-1306042 GUGCACUGUGACGUCCACUUCGG 1452 AD-1306069 GUGUGCAGGAACUGGGAGCAGGA 1453 AD-1306099 UUCAAGAUGUGCUACAACUACAG 1454 AD-1306132 CUCUGCUGCAGUGACGACCACUG 1455 AD-1306144 ACCGACCACAGAGCUGGAGACGG 1456 AD-1306168 CCACCCAGGCCCUGUUCUCAACG 1457 AD-1306198 CCCUCUCAGAAGGACUGACAUCC 1458 AD-1306200 CCCAGAUACACAAGCACCCUUGG 1459 AD-1306235 AGGCUCCACAGAACCCACUGUCC 1460 AD-1306254 UCCACCCUUCCAACACGCUCAGC 1461 AD-1306278 CCCAACAACAAUGGCAACCUCCA 1462 AD-1306324 GCACCGCUUCCAAAGAGCCGCUG 1463 AD-1306359 CGCCAACACUCACGAGCGAGCUG 1464 AD-1306382 UCCACCUCUCAGGCCGAGACCAG 1465 AD-1306406 GCCCAGGACAGAGACGACAAUGA 1466 AD-1306411 CCCUUGACUAACACCACCACCAG 1467 AD-1306442 CGCUGUCAACCGAAGUGUGAGUG 1468 AD-1306478 GACGUGGACUUCCCAACCUCAGG 1469 AD-1306483 GGACAUGGAAACUUUUGAAAACA 1470 AD-1306514 GGGCACCAAAGAGCAUAGAGUGC 1471 AD-1306535 GAGGUAAGCAUCGACCAGGUCGG 1472 AD-1306563 UGCUGACCUGCAGCCUGGAGACG 1473 AD-1306575 CCUGCAAGAACGAAGACCAGACA 1474 AD-1306603 GUUCAACAUGUGCUUCAACUACA 1475 AD-1306627 CGUGCGUGUGCUUUGCUGUGACG 1476 AD-1306673 ACCUCCACCCUGAGAACAGCUCC 1477 AD-1306676 CCUCCCAAAGUGCUGACCACCAC 1478 AD-1306698 UCUUCCUCCCUGGGCACCACCUG 1479 AD-1306717 AGUGCCAACUACCACAACCACGG 1480 AD-1307191 ACCACACCCACAACCAGAGGCUC 1481 AD-1307354 ACGACCUGGAUCCUCACAAAGCC 1482 AD-1308029 AGCUACCAGCGUUACACCCAUCC 1483 AD-1308183 GACCACAACAGCCACUACGACUG 1484 AD-1308294 ACCAGCUCCAAAGCCACUCCCUC 1485 AD-1334092 CCGCCCUUCCAGCACUGAGAAGC 1486 AD-1308403 CGCCUAUCACAGACCACCACACC 1487 AD-1308420 ACGGCCACCAUGUCCACAGCCAC 1488 AD-1308440 ACCCUCCUCCACUCCAGAGACUG 1489 AD-1334093 CCCACACCUCCACAGUGCUUACC 1490 AD-1308488 CCCAGGAACAGCUCACACUACCA 1491 AD-1308555 CCUCCAGUGUGGAUCAGCACAAC 1492 AD-1306730 AACUCCUGGGACAACUCCCAUCC 1493 AD-1306746 ACCAGCAACACAGUGACUCCCUC 1494 AD-1306753 CCAGUGCCGAACACCAUGGCCAC 1495 AD-1306772 ACGGUGACUUCCCACACCCUAGC 1496 AD-1306796 CUCGACUCCAGCCCUUUCCAGCC 1497 AD-1306820 CCUAGCAGCAGAACCACCGAGUC 1498 AD-1306844 CAGCUCACACUACCAAAGUGCUG 1499 AD-1306879 GCACGCUUCCAGUGUGGAUCAGC 1500 AD-1306887 CACACCCACAACCAGAGGUUCCA 1501 AD-1306916 ACCACGGUGGUGACCAUGGGCUG 1502 AD-1307191 ACCACACCCACAACCAGAGGCUC 1481 AD-1307212 ACACCGCCACAGUGCUGACCACC 1503 AD-1307378 AGCCACUACGACUGAGUCCACUG 1504 AD-1307392 CAGCUCCAAAGCCACUCCCUUCU 1505 AD-1307516 AACUCCAGGGACAACACCUAUCC 1506 AD-1307551 ACCAGCAGCACAGUGACUCCCUC 1507 AD-1307575 UCUGCCCUAGGGACCACCCACAC 1508 AD-1307600 ACCACACACGGGCGAUCCCUGUC 1509 AD-1307617 ACAGCCUGGACUUCGGCCACCUC 1510 AD-1307654 CCACCCACAUCACAGAGCCUUCC 1511 AD-1307666 AGCAACCACCGGUACCACCCAGC 1512 AD-1307754 ACACCCAGCAAGACCCGCACCUC 1513 AD-1307805 GAGUGGCUGGACUACAGCUACCC 1514 AD-1307812 CUUUGACACCUACUCCAACAUCC 1515 AD-1307837 AGUUGGGCCAGGUCGUGGAAUGC 1516 AD-1307860 AGCCUGGACUUUGGCCUGGUCUG 1517 AD-1307893 AAGAUGUGCUUCAACUAUGAAAU 1518 AD-1307917 CGUGUGUUCUGCUGCAACUACGG 1519 AD-1307934 ACCAGCUCUACGGCCAUGCCCUC 1520 AD-1308171 GGAUCCUCACAGAGCUGACCACA 1521 AD-1308216 AGCCGAGCACUACAGCCACCGUG 1522 AD-1308250 CUCCUCCACCCAGGCAACUGCUG 1523 AD-1308273 ACGGCCACGACACCCACAGUCAC 1524 AD-1334092 CCGCCCUUCCAGCACUGAGAAGC 1486 AD-1308369 CCCACAGCUACCAGCUUUACAGC 525 AD-1308403 CGCCUAUCACAGACCACCACACC 1487 AD-1308420 ACGGCCACCAUGUCCACAGCCAC 1488 AD-1308440 ACCCUCCUCCACUCCAGAGACUG 1489 AD-1308463 UCCACACCUCCACAGUGCUUACC 1526 AD-1308520 CUACCACAACCACGGGCUUCACA 1527 AD-1308605 CUGACCACCACCACCACAACUGU 1528 AD-1308629 GCCACUGGUUCUAUGGCAACACC 1529 AD-1308652 CUCCUCUAGCACACAGACCAGUG 1530 AD-1308673 CACCACGGCCACUACGAUCACGG 1531 AD-1309037 CAGGGACCACCUGGAUCCUCACA 1532 AD-1306916 ACCACGGUGGUGACCAUGGGCUG 1502 AD-1306975 AGCAGCCACUACGACCGCAACCA 1533 AD-1307006 CCUCCCAAAGUGCUGACCAGCAC 1534 AD-1307011 CCCUCCUUCACCCUUGGGACCAC 1535 AD-1307040 AAGGGCCACCAGUUCCAUGUCCA 1536 AD-1307063 CCCAGCACUACAGCCACCGUGAC 1537 AD-1307092 GCACCCUCAAAGUGCUGACCAGC 1538 AD-1307111 ACCACACCCACAGUCAUCAGCUC 1539 AD-1307137 CACACCCACAGCUACCAGCGUUA 1540 AD-1307167 CCUCCUCUACUCCAGAGACUGUC 1541 AD-1307174 CCACAGUGCUUACCACCACGACC 1542 AD-1307191 ACCACACCCACAACCAGAGGCUC 1481 AD-1307212 ACACCGCCACAGUGCUGACCACC 1503 AD-1307222 CGGCCACUACGAUCACAGCCACC 1543 AD-1307242 AACUCCAGGGACAACUCCCAUCC 1544 AD-1307288 CCCAGCAGCAACCACCAGUACCA 1545 AD-1307310 ACCUCCAGGACCACAGCCACAGC 1546 AD-1307346 UACGGCCACGCCCUCCUCAACUC 1547 AD-1307363 AUCCUCACAAAGCUGACCACAAC 1548 AD-1307378 AGCCACUACGACUGAGUCCACUG 1504 AD-1307392 CAGCUCCAAAGCCACUCCCUUCU 1505 AD-1307431 CCUCCUCCACUCCAGAGACUGCC 1549 AD-1307456 CUCCACAGUGCUUACCACCACGG 1550 AD-1307516 AACUCCAGGGACAACACCUAUCC 1506 AD-1307551 ACCAGCAGCACAGUGACUCCCUC 1507 AD-1307575 UCUGCCCUAGGGACCACCCACAC 1508 AD-1307590 AACACCACGGCCACCACACACGG 1551 AD-1307600 ACCACACACGGGCGAUCCCUGUC 1509 AD-1307617 ACAGCCUGGACUUCGGCCACCUC 1510 AD-1307654 CCACCCACAUCACAGAGCCUUCC 1511 AD-1307677 GUACCACCCAGCACUCGACUCCA 1552 AD-1307706 UCCAGCCCUCACCCUAGCAGCAG 1553 AD-1307754 ACACCCAGCAAGACCCGCACCUC 1513 AD-1307775 CCAUAACCACGGUGGUGACCACG 1554 AD-1307805 GAGUGGCUGGACUACAGCUACCC 1514 AD-1307812 CUUUGACACCUACUCCAACAUCC 1515 AD-1307837 AGUUGGGCCAGGUCGUGGAAUGC 1516 AD-1307860 AGCCUGGACUUUGGCCUGGUCUG 1517 AD-1307893 AAGAUGUGCUUCAACUAUGAAAU 1518 AD-1307917 CGUGUGUUCUGCUGCAACUACGG 1519 AD-1307934 ACCAGCUCUACGGCCAUGCCCUC 1520 AD-1307994 CACCAGUUCCAAAGCCACUUCCU 1555 AD-1308057 CUCCCAGAACAGACCACCACACC 1556 AD-1308098 ACCUCCACAGUGCUGACCACGAA 1557 AD-1308142 ACGACCUGGAUCCUCACAGAGCU 1558 AD-1308171 GGAUCCUCACAGAGCUGACCACA 1521 AD-1308216 AGCCGAGCACUACAGCCACCGUG 1522 AD-1308250 CUCCUCCACCCAGGCAACUGCUG 1523 AD-1308273 ACGGCCACGACACCCACAGUCAC 1524 AD-1308305 AGCCACUCCCUCCUCCAGUCCAG 1559 AD-1334092 CCGCCCUUCCAGCACUGAGAAGC 1486 AD-1308369 CCCACAGCUACCAGCUUUACAGC 1525 AD-1308403 CGCCUAUCACAGACCACCACACC 1487 AD-1308422 GGCCACCAUGUCCACAGCCACAC 1560 AD-1308488 CCCAGGAACAGCUCACACUACCA 1491 AD-1308512 AGUGCCGACUACCACAACCACGG 1561 AD-1308555 CCUCCAGUGUGGAUCAGCACAAC 1492 AD-1308569 ACCACACCCACAACCAGUGGCUC 1562 AD-1308605 CUGACCACCACCACCACAACUGU 1528 AD-1308629 GCCACUGGUUCUAUGGCAACACC 1529 AD-1308652 CUCCUCUAGCACACAGACCAGUG 1530 AD-1308673 CACCACGGCCACUACGAUCACGG 1531 AD-1308759 UCCAAGGACUGCAACCACCCUUC 1563 AD-1308782 CAGUGCUGACAAGCACAGCCACC 1564 AD-1308808 UCCACAGCUACCAGCUUUACACC 1565 AD-1334094 GGCCACCAUGUCCACAAUCCACC 1566 AD-1309007 AGCCACUACAACUGCAGCCACUG 1567 AD-1309037 CAGGGACCACCUGGAUCCUCACA 1532 AD-1309039 GGGACCACCUGGAUCCUCACAGA 1568 AD-1307953 AGCCACUACGACUGCAUCCACUG 1569 AD-1307967 CCCUCCCAAAGUGCUGACCAGCA 1570 AD-1307994 CACCAGUUCCAAAGCCACUUCCU 1555 AD-1308024 UCCACAGCUACCAGCGUUACACC 1571 AD-1308057 CUCCCAGAACAGACCACCACACC 1556 AD-1308098 ACCUCCACAGUGCUGACCACGAA 1557 AD-1308133 AAGGGCCACCAGUUCCACGUCCA 1572 AD-1308171 GGAUCCUCACAGAGCUGACCACA 1521 AD-1308180 GCUGACCACAACAGCCACUACGA 1573 AD-1308216 AGCCGAGCACUACAGCCACCGUG 1522 AD-1308250 CUCCUCCACCCAGGCAACUGCUG 1523 AD-1308263 UGUGAGCACCACGGCCACGACAC 1574 AD-1308294 ACCAGCUCCAAAGCCACUCCCUC 1485 AD-1308317 CUCCAGUCCAGGGACUGCAACUG 1575 AD-1308344 UCCAGCACUGAGAAGCACAGCCA 1576 AD-1308369 CCCACAGCUACCAGCUUUACAGC 1525 AD-1308403 CGCCUAUCACAGACCACCACACC 1487 AD-1308420 ACGGCCACCAUGUCCACAGCCAC 1488 AD-1308440 ACCCUCCUCCACUCCAGAGACUG 1489 AD-1308463 UCCACACCUCCACAGUGCUUACC 1526 AD-1308488 CCCAGGAACAGCUCACACUACCA 1491 AD-1308512 AGUGCCGACUACCACAACCACGG 1561 AD-1308555 CCUCCAGUGUGGAUCAGCACAAC 1492 AD-1308566 ACCACCACACCCACAACCAGUGG 1577 AD-1308595 ACCGCCAGAGUGCUGACCACCAC 1578 AD-1308629 GCCACUGGUUCUAUGGCAACACC 1529 AD-1308652 CUCCUCUAGCACACAGACCAGUG 1530 AD-1308673 CACCACGGCCACUACGAUCACGG 1531 AD-1308702 UCCAGGGACAACACCCAUCACCC 1579 AD-1308733 AGCUCCAAAGCCACUUCCUCCUC 1580 AD-1308759 UCCAAGGACUGCAACCACCCUUC 1563 AD-1308782 CAGUGCUGACAAGCACAGCCACC 1564 AD-1308783 AGUGCUGACAAGCACAGCCACAA 1581 AD-1308808 UCCACAGCUACCAGCUUUACACC 1565 AD-1308818 UCCUCCACCCUGUGGACCACGUG 1582 AD-1308845 GUCCCAGCACAGACCACCACACC 1583 AD-1308868 AUGUCCACCAUGUCCACAAUCCA 1584 AD-1334094 GGCCACCAUGUCCACAAUCCACC 1566 AD-1308890 CACCUCCUCUACUCCAGAGACCA 1585 AD-1308912 CCCACACCUCCACAGUGCUGACC 1586 AD-1308935 ACCACAGCCACCAUGACAAGGGC 1587 AD-1308963 AUUCCACGGCCACACCCUCCUCC 1588 AD-1308975 ACGACCCGGAUCCUCACUGAGCU 1589 AD-1308998 GACCACAACAGCCACUACAACUG 1590 AD-1309007 AGCCACUACAACUGCAGCCACUG 1567 AD-1309026 ACUGGAUCCACGGCCACCCUGUC 1591 AD-1309037 CAGGGACCACCUGGAUCCUCACA 1532 AD-1309039 GGGACCACCUGGAUCCUCACAGA 1568 AD-1309042 ACCACCUGGAUCCUCACAGAGCC 1592 AD-1309069 ACUAUAGCCACCGUGAUGGUGCC 1593 AD-1309105 CUCCACUCUGGGAACAGCUCACA 1594 AD-1309124 ACCAUGGCCACUAUGCCCACAGC 1595 AD-1309148 ACUGCCUCCACGGUUCCCAGCUC 1596 AD-1309167 AGCCUGCCAACCUUCAGCGUGUC 1597 AD-1309192 CUGUGUCCUCCUCAGUCCUCACC 1598 AD-1309216 CCCAGCUCCCACUUCUCUACUCC 1599 AD-1309250 GGGCAUUUGGACAGUUUUUCUCG 1600 AD-1309259 GGGAAGUCAUCUACAAUAAGACC 1601 AD-1309287 GGCUGCCAUUUCUACGCAGUGUG 1602 AD-1309315 AGCACUGUGACAUUGACCGCUUC 1603 AD-1309337 GCUGUGACAAUGCCAUCCCUCUC 1604 AD-1309379 GGACCCUGGAGAACUGCACGGUG 1605 AD-1309406 GCGUGGGUGACAACCGUGUCGUC 1606 AD-1309433 UGGACCCAAAGCCUGUGGCCAAC 1607 AD-1309459 ACCUGCGUGAACAAGCACCUGCC 1608 AD-1309482 CAUCAAAGUGUCGGACCCGAGCC 1609 AD-1309503 CCCUGUGACUUCCACUAUGAGUG 1610 AD-1309526 CGAGUGCAUCUGCAGCAUGUGGG 1611 AD-1309535 CUCCCACUAUUCCACCUUUGACG 1612 AD-1309567 GCACCUAUGUCCUCAUGAGAGAG 1613 AD-1309595 UGCACGCUUUGGGAAUCUCAGCC 1614 AD-1309621 ACCUGGACAACCACUACUGCACG 1615 AD-1309647 GCCCUCAGCAUCCACUACAAGUC 1616 AD-1309678 UCGUCCUCACUGUCACCAUGGUG 1617 AD-1309720 UGAUCCUGUUUGACCAAAUUCCG 1618 AD-1309747 GCAGCGGUUUCAGCAAGAACGGC 1619 AD-1309780 CCAUGCGUGUGGACAUUCCUGCC 1620 AD-1309804 GCGUGAGCGUCACCUUCAAUGGC 1621 AD-1309844 ACAGCCUCUUCCACAACAACACC 1622 AD-1309872 GGCACCUGCACCAACAACCAGAG 1623 AD-1309900 ACUGUCUCCAGCGGGACGGAACC 1624 AD-1309926 GCCGCCAGUUGCAAGGACAUGGC 1625 AD-1309945 CCCGACAGCAGAAAGGAUGGCUG 1626 AD-1309969 AGCCGCUCUGUGAUCUGAUGCUG 1627 AD-1309993 GCCAGGUCUUUGCUGAGUGCCAC 1628 AD-1310022 AACGCCUGCAUCAGCGACCACUG 1629 AD-1310059 UGGAGGCUUACGCAGAGCUCUGC 1630 AD-1310068 GGAGUGUGCAGUGACUGGCGAGG 1631 AD-1310124 CCCACCAAAGUGUACAAGCCAUG 1632 AD-1310146 ACCUGCAACUCUAGGAACCAGAG 1633 AD-1310192 AGGACCAGAUCCUCUUCAACGCA 1634 AD-1310215 CACAUGGGCAUCUGCGUGCAGGC 1635 AD-1310234 CCCGAUGGGUUUCCUAAAUUUCC 1636 AD-1310249 UGGGUCAGCAACUGCCAGUCCUG 1637 AD-1310280 ACGAGGGUUCAGUGUCGGUGCAG 1638 AD-1310310 UCCCGGCUUCGUAACCGUGACCA 1639 AD-1310328 UGCGUGUGCAACACAACCACCUG 1640 AD-1310345 AGGGCAGGAGUCCAUCUGCACCC 1641 AD-1310378 GACUGCUGUCCCACCUUCCGCUG 1642 AD-1310405 CCUCAGCUGUGUUCGUACAAUGG 1643 AD-1310422 GGUUGGUGCAACCUUCCCAGGCG 1644 AD-1310443 CUUCCCUGCCACAUGUGUACCUG 1645 AD-1310466 GGACCCAACGGUGCAAUGUCAGG 1646 AD-1310493 UGCCUGCAACAAUACUACCUGUC 1647 AD-1310497 CCAGGGCUUUGAGUACAAGAGAG 1648 AD-1310552 CCAGUCCAGCUGAAUGAAACCUG 1649 AD-1310576 GUCAACAGCCAUGUGGACAACUG 1650 AD-1310599 CACCGUGUACCUCUGUGAGGCUG 1651 AD-1310622 AGGGUGGAGUCCAUUUGCUGACC 1652 AD-1310637 UCCUGCCCAGAUGUGUCCAGCUG 1653 AD-1310665 CUGCUGCUACUCCUGUGAGGAGG 1654 AD-1310689 CUCCUGUCAAGUCCGCAUCAACA 1655 AD-1310714 ACCAUCCUGUGGCACCAGGGCUG 1656 AD-1310741 ACCGAGGUCAACAUCACCUUCUG 1657 AD-1310762 AGCGUCCAAGUACUCAGCAGAGG 1658 AD-1310790 GCCAUGCAGCACCAGUGCACCUG 1659 AD-1310845 CCCUUGCACUGUCCUAACGGCUC 1660 AD-1310873 UCCUGCACACCUACACCCACGUG 1661 AD-1310907 CUGCACGCCCUUCUGUGUCCCUG 1662 AD-1310938 CCACUGCUGUCUGAGAACGUUCU 1663 AD-1310950 CCCAUGCUCUGUCCACCUGGAGC 1664 AD-1310979 GUGCAUUGUCUGAUCAUGAAAAC 1665 AD-1311016 AGGGCGCCACUCAGGAGUCCUAC 1666 AD-1311052 CCCUCCCUGAUGUCACUGGGACG 1667 AD-1311075 CCCUGGAACAAACUAAGCAUGUG 1668 AD-1311115 AGCACGGAUUCCAGCUGGCCACG 1669 AD-1311126 CAGACAGGCUGGUCCAGGCAAGG 1670 AD-1311154 UGCUGCCAGGAAGCUGCGACAGG 1671 AD-1311185 GCCUGCUGCAGGGUAACUCAGGG 1672 AD-1311214 UCGCAACGGCCAGGUCAGAGAGG 1673 AD-1311231 AGCCCAGUUUUGCAAAUAAACCC 1674

TABLE 4 Unmodified Sense and Antisense Strand MUC5B dsRNA Sequences Duplex SEQ ID SEQ ID Start Site(s) in Name Sense Sequence 5′ to 3′ NO: Antisense Sequence 5′ to 3′ NO: NM_002458.3 AD-1334097 UUGGCUCUGGCGGCCAUGCUA 1675 UAGCAUGGCCGCCAGAGCCAACA 1999    90 AD-1334098 CUGGGAGAAUGCAGGGCACAA 1676 UUGUGCCCUGCAUUCUCCCAGCU 2000   152 AD-1334099 GCGCGUGAGCUUUGUUCCACA 1677 UGUGGAACAAAGCUCACGCGCCG 2001   209 AD-1334100 UGGGCGGGUGUGCAGCACCUA 1678 UAGGUGCUGCACACCCGCCCAUU 2002   275 AD-1334101 CCACUACAAGACCUUCGACGA 1679 UCGUCGAAGGUCUUGUAGUGGAA 2003   305 AD-1334102 CCUUUGCAACUACGUGUUCUA 1680 UAGAACACGUAGUUGCAAAGGCC 2004   347 AD-1334103 GAGGACUUCAACGUCCAGCUA 1681 UAGCUGGACGUUGAAGUCCUCGU 2005   390 AD-1334104 ACCCGUGUUGUCAUCAAGGCA 1682 UGCCUUGAUGACAACACGGGUGA 2006   444 AD-1334105 GGCUCCGUCCUCAUCAAUGGA 1683 UCCAUUGAUGAGGACGGAGCCGU 2007   492 AD-1334106 GCUGCCUUACAGCCGCACUGA 1684 UCAGUGCGGCUGUAAGGCAGCUC 2008   524 AD-1334107 GACUACAUCAAGGUCAGCAUA 1685 UAUGCUGACCUUGAUGUAGUCCC 2009   567 AD-1334108 GCUGACAUUCCUGUGGAACGA 1686 UCGUUCCACAGGAAUGUCAGCAC 2010   596 AD-1334109 CUGGAUCCCAAAUACGCCAAA 1687 UUUGGCGUAUUUGGGAUCCAGCU 2011   639 AD-1334110 GCCUUCAACGAGUUCUAUGCA 1688 UGCAUAGAACUCGUUGAAGGCCG 2012   699 AD-1334111 AACCUGCAGAAGUUGGAUGGA 1689 UCCAUCCAACUUCUGCAGGUUCC 2013   753 AD-1334112 UGCACGGACGAGGAGGGCAUA 1690 UAUGCCCUCCUCGUCCGUGCAGU 2014   819 AD-1334113 UUUGCGGAGUGCCACGCACUA 1691 UAGUGCGUGGCACUCCGCAAAGG 2015   867 AD-1334114 GACAGCACUGCGUACCUGGCA 1692 UGCCAGGUACGCAGUGCUGUCCA 2016   891 AD-1334115 GCCACCUUUGUGGAAUACUCA 1693 UGAGUAUUCCACAAAGGUGGCAC 2017   954 AD-1334116 CUGGAGGUGCCCUGAGCUCUA 1694 UAGAGCUCAGGGCACCUCCAGUU 2018  1010 AD-1334117 CUCAACAUGCAGCACCAGGAA 1695 UUCCUGGUGCUGCAUGUUGAGGG 2019  1047 AD-1334118 ACCCUGCACGGACACCUGCUA 1696 UAGCAGGUGUCCGUGCAGGGUGA 2020  1076 AD-1334119 GGACCACUGUGUGGACGGCUA 1697 UAGCCGUCCACACAGUGGUCCUC 2021  1124 AD-1334120 GCUGGAUGACAUCACGCACUA 1698 UAGUGCGUGAUGUCAUCCAGCAC 2022  1166 AD-1334121 CUCCUUCAACACCACCUGCAA 1699 UUGCAGGUGGUGUUGAAGGAGGU 2023  1250 AD-1334122 CUAUGGCAGUGCCAGGACCUA 1700 UAGGUCCUGGCACUGCCAUAGCC 2024  1293 AD-1334123 ACCUAUGAUGAGAAACUCUAA 1701 UUAGAGUUUCUCAUCAUAGGUGG 2025  1359 AD-1334124 AGCUACGUUCUGUCCAAGAAA 1702 UUUCUUGGACAGAACGUAGCUGC 2026  1398 AD-1334125 GACAGCAGCUUCACCGUGCUA 1703 UAGCACGGUGAAGCUGCUGUCGG 2027  1425 AD-1334126 GGACAACGAGAACUGCCUGAA 1704 UUCAGGCAGUUCUCGUUGUCCGU 2028  1472 AD-1334127 UCCUCAACUCCAUCUACACGA 1705 UCGUGUAGAUGGAGUUGAGGAAC 2029  1558 AD-1334128 GCCAACAUCACCCUGUUCACA 1706 UGUGAACAGGGUGAUGUUGGCUG 2030  1596 AD-1334129 UCGAGCUUCUUCAUCGUGGUA 1707 UACCACGAUGAAGAAGCUCGAGG 2031  1620 AD-1334130 GCCACUCAUGCAGGUGUUUGA 1708 UCAAACACCUGCAUGAGUGGCAC 2032  1679 AD-1334131 GGGAACUUCAACCAGAACCAA 1709 UUGGUUCUGGUUGAAGUUCCCAC 2033  1743 AD-1334132 UGACGACUUCACGGCCCUCAA 1710 UUGAGGGCCGUGAAGUCGUCAGC 2034  1766 AD-1334133 AGCCUUCGCCAACACCUGGAA 1711 UUCCAGGUGUUGGCGAAGGCUGC 2035  1811 AD-1334134 CCAGGAACAGCUUUGAGGACA 1712 UGUCCUCAAAGCUGUUCCUGGCA 2036  1855 AD-1334135 GUGGAGAAUGAGAACUACGCA 1713 UGCGUAGUUCUCAUUCUCCACAC 2037  1890 AD-1334136 CAACAGUGCCUUCUCGCGCUA 1714 UAGCGCGAGAAGGCACUGUUGGG 2038  1940 AD-1334137 CUUCCACUCGAACUGCAUGUA 1715 UACAUGCAGUUCGAGUGGAAGGG 2039  1985 AD-1334138 CACCUGCAACUGUGAGCGGAA 1716 UUCCGCUCACAGUUGCAGGUGUC 2040  2009 AD-1334139 CCUCCUAUGUGCACGCCUGUA 1717 UACAGGCGUGCACAUAGGAGGAC 2041  2056 AD-1334140 GGCGUACAGCUCAGCGACUGA 1718 UCAGUCGCUGAGCUGUACGCCCU 2042  2085 AD-1334141 ACCAAGUACAUGCAGAACUGA 1719 UCAGUUCUGCAUGUACUUGGUGC 2043  2121 AD-1334142 UACGCCUACGUGGUGGAUGCA 1720 UGCAUCCACCACGUAGGCGUAGC 2044  2157 AD-1334143 GCAGCGUUUCCUUCGUGCCUA 1721 UAGGCACGAAGGAAACGCUGCAG 2045  2221 AD-1334144 GCACCUUCCUCAAUGACGCGA 1722 UCGCGUCAUUGAGGAAGGUGCCC 2046  2266 AD-1334145 CUGGAGAGGUGGUGCACGACA 1723 UGUCGUGCACCACCUCUCCAGGA 2047  2344 AD-1334146 CCGUGUGUUCAUGUACGGGUA 1724 UACCCGUACAUGAACACACGGCG 2048  2371 AD-1334147 CCUCUCUGCAGAAAAGCACAA 1725 UUGUGCUUUUCUGCAGAGAGGCU 2049  2413 AD-1334148 GGACUGCAGCAACAGCUCGGA 1726 UCCGAGCUGUUGCUGCAGUCCAG 2050  2459 AD-1334149 CUGUUUCAGCACACACUGCGA 1727 UCGCAGUGUGUGCUGAAACAGCC 2051  2531 AD-1334150 CUGCAUUGCCGAGGAGGACUA 1728 UAGUCCUCCUCGGCAAUGCAGCC 2052  2600 AD-1334151 CACCUACAAGCCUGGAGAGAA 1729 UUCUCUCCAGGCUUGUAGGUGGC 2053  2642 AD-1334152 CGACUGCAACACCUGCACCUA 1730 UAGGUGCAGGUGUUGCAGUCGAC 2054  2672 AD-1334153 GAACCGGAGGUGGGAGUGCAA 1731 UUGCACUCCCACCUCCGGUUCCU 2055  2696 AD-1334154 UGGCCACUUCAUCACCUUUGA 1732 UCAAAGGUGAUGAAGUGGCCAUC 2056  2756 AD-1334155 CGAUCGCUACAGCUUUGAAGA 1733 UCUUCAAAGCUGUAGCGAUCGCC 2057  2780 AD-1334156 GCUGCGAGUACAUCUUGGCCA 1734 UGGCCAAGAUGUACUCGCAGCUG 2058  2803 AD-1334157 CUUCCGCAUCGUCACCGAGAA 1735 UUCUCGGUGACGAUGCGGAAGGU 2059  2858 AD-1334158 GCCAUCAAGCUCUUCGUGGAA 1736 UUCCACGAAGAGCUUGAUGGCCU 2060  2916 AD-1334159 UACGAGCUGAUCCUCCAAGAA 1737 UUCUUGGAGGAUCAGCUCGUAGC 2061  2940 AD-1334160 GACCUUUAAGGCGGUGGCGAA 1738 UUCGCCACCGCCUUAAAGGUCCC 2062  2963 AD-1334161 ACCCUACAAGAUACGCUACAA 1739 UUGUAGCGUAUCUUGUAGGGUGG 2063  3002 AD-1334162 UUCCUGGUCAUCGAGACCCAA 1740 UUGGGUCUCGAUGACCAGGAAGA 2064  3030 AD-1334163 CCAGCGUGUUCAUCCGACUGA 1741 UCAGUCGGAUGAACACGCUGGUC 2065  3079 AD-1334164 CAGGACUACAAGGGCAGGGUA 1742 UACCCUGCCCUUGUAGUCCUGGU 2066  3102 AD-1334165 GGGAACUUCGACGACAAUGCA 1743 UGCAUUGUCGUCGAAGUUCCCGC 2067  3135 AD-1334166 CAAUGACUUUGCCACGCGUAA 1744 UUACGCGUGGCAAAGUCAUUGAU 2068  3158 AD-1334167 GCACUGGAGUUUGGGAACAGA 1745 UCUGUUCCCAAACUCCAGUGCGU 2069  3198 AD-1334168 GGCCCAGAAGCAGUGCAGCAA 1746 UUGCUGCACUGCUUCUGGGCCCA 2070  3296 AD-1334169 CCAGGUUGACUCCACCAAGUA 1747 UACUUGGUGGAGUCAACCUGGGA 2071  3350 AD-1334170 CGAGGCCUGCGUGAACGACGA 1748 UCGUCGUUCACGCAGGCCUCGUA 2072  3374 AD-1334171 ACUGCGAGUGUUUCUGCACGA 1749 UCGUGCAGAAACACUCGCAGUCG 2073  3418 AD-1334172 UGUGUGUGUCCUGGCGGACUA 1750 UAGUCCGCCAGGACACACACAGG 2074  3478 AD-1334173 UGUUCUGUGACUUCUACAACA 1751 UGUUGUAGAAGUCACAGAACAAG 2075  3514 AD-1334174 UGUGAGUGGCACUACCAGCCA 1752 UGGCUGGUAGUGCCACUCACAGC 2076  3546 AD-1334175 GCUGCUACCCGAAGUGCCCAA 1753 UUGGGCACUUCGGGUAGCAGCCU 2077  3640 AD-1334176 AGCCCUUCUUCAAUGAGGACA 1754 UGUCCUCAUUGAAGAAGGGCUGG 2078  3667 AD-1334177 AAGUGCGUGGCCCAGUGUGGA 1755 UCCACACUGGGCCACGCACUUCA 2079  3693 AD-1334178 CUACGACAAGGACGGAAACUA 1756 UAGUUUCCGUCCUUGUCGUAGCA 2080  3716 AD-1334179 UGACGUCGGUGCAAGGGUCCA 1757 UGGACCCUUGCACCGACGUCAUA 2081  3740 AD-1334180 CCAGAGCUGUAACUGCACACA 1758 UGUGUGCAGUUACAGCUCUGGCA 2082  3776 AD-1334181 CAGUGCGCUCACAGCCUUGAA 1759 UUCAAGGCUGUGAGCGCACUGGA 2083  3807 AD-1334182 CUGCACCUAUGAGGACAGGAA 1760 UUCCUGUCCUCAUAGGUGCAGGU 2084  3836 AD-1334183 CAGGACGUCAUCUACAACACA 1761 UGUGUUGUAGAUGACGUCCUGGU 2085  3867 AD-1334184 CGCCUGCUUGAUCGCCAUCUA 1762 UAGAUGGCGAUCAAGCAGGCGCC 2086  3902 AD-1334185 ACCAUCAUCAGGAAGGCUGUA 1763 UACAGCCUUCCUGAUGAUGGUGC 2087  3936 AD-1334186 CACAACGCCAUUCACCUUCAA 1764 UUGAAGGUGAAUGGCGUUGUGGC 2088  3977 AD-1334187 UCCACCGUGUGUGUCCGCGAA 1765 UUCGCGGACACACACGGUGGAGA 2089  4044 AD-1334188 UCCAGCUGGUACAAUGGGCAA 1766 UUGCCCAUUGUACCAGCUGGACC 2090  4077 AD-1334189 CGGAGACUUUGAGACGUUUGA 1767 UCAAACGUCUCAAAGUCUCCGCC 2091  4121 AD-1334190 GAGGGUACCAGGUAUGCCCUA 1768 UAGGGCAUACCUGGUACCCUCUC 2092  4156 AD-1334191 CUGGCUGACAUCGAGUGCCGA 1769 UCGGCACUCGAUGUCAGCCAGCA 2093  4179 AD-1334192 CUUCCCGACAUGCCGCUGGAA 1770 UUCCAGCGGCAUGUCGGGAAGCU 2094  4209 AD-1334193 CAGGUGGACUGUGACCGCAUA 1771 UAUGCGGUCACAGUCCACCUGCU 2095  4242 AD-1334194 CGCCAACAGCCAACAGAGUCA 1772 UGACUCUGUUGGCUGUUGGCGCA 2096  4277 AD-1334195 UCUGUCACGACUACGAGCUGA 1773 UCAGCUCGUAGUCGUGACAGAGC 2097  4303 AD-1334196 UCUCUGCUGCGAAUACGUGCA 1774 UGCACGUAUUCGCAGCAGAGAAC 2098  4328 AD-1334197 CACGGAGCCUGCUGUGCCUAA 1775 UUAGGCACAGCAGGCUCCGUGCU 2099  4403 AD-1334198 AGACCACAGCAACCGAAAAGA 1776 UCUUUUCGGUUGCUGUGGUCUGG 2100  4432 AD-1334199 CACCUCGCAGACUGGGUCCAA 1777 UUGGACCCAGUCUGCGAGGUGAG 2101  4496 AD-1334200 ACAGAGUGGUUUGAUGAGGAA 1778 UUCCUCAUCAAACCACUCUGUCC 2102  4584 AD-1334201 GACGUUGAGUCCUACGAUAAA 1779 UUUAUCGUAGGACUCAACGUCCC 2103  4632 AD-1334202 GGCCGCUGGAGGGCACUUAUA 1780 UAUAAGUGCCCUCCAGCGGCCCU 2104  4658 AD-1334203 CAGCCUAAGGACAUAGAGUGA 1781 UCACUCUAUGUCCUUAGGCUGCU 2105  4683 AD-1334204 AACUGGACCCUGGCACAGGUA 1782 UACCUGUGCCAGGGUCCAGUUGG 2106  4722 AD-1334205 GUGCACUGUGACGUCCACUUA 1783 UAAGUGGACGUCACAGUGCACCU 2107  4752 AD-1334206 GUGCAGGAACUGGGAGCAGGA 1784 UCCUGCUCCCAGUUCCUGCACAC 2108  4781 AD-1334207 CGUCUUCAAGAUGUGCUACAA 1785 UUGUAGCACAUCUUGAAGACGCC 2109  4805 AD-1334208 CUGCUGCAGUGACGACCACUA 1786 UAGUGGUCGUCACUGCAGCAGAG 2110  4844 AD-1334209 CGACCACAGAGCUGGAGACGA 1787 UCGUCUCCAGCUCUGUGGUCGGU 2111  4891 AD-1334210 GCCCUGUUCUCAACGCCGCAA 1788 UUGCGGCGUUGAGAACAGGGCCU 2112  4932 AD-1334211 CCUCUCAGAAGGACUGACAUA 1789 UAUGUCAGUCCUUCUGAGAGGGU 2113  5012 AD-1334212 CAGAUACACAAGCACCCUUGA 1790 UCAAGGGUGCUUGUGUAUCUGGG 2114  5036 AD-1334213 GCUCCACAGAACCCACUGUCA 1791 UGACAGUGGGUUCUGUGGAGCCU 2115  5092 AD-1334214 CACCCUUCCAACACGCUCAGA 1792 UCUGAGCGUGUUGGAAGGGUGGA 2116  5129 AD-1334215 CAACAACAAUGGCAACCUCCA 1793 UGGAGGUUGCCAUUGUUGUUGGG 2117  5212 AD-1334216 CGCUUCCAAAGAGCCGCUGAA 1794 UUCAGCGGCUCUUUGGAAGCGGU 2118  5261 AD-1334217 GCGCCAACACUCACGAGCGAA 1795 UUCGCUCGUGAGUGUUGGCGCCA 2119  5292 AD-1334218 GUCCACCUCUCAGGCCGAGAA 1796 UUCUCGGCCUGAGAGGUGGACAG 2120  5315 AD-1334219 CAGGACAGAGACGACAAUGAA 1797 UUCAUUGUCGUCUCUGUCCUGGG 2121  5345 AD-1334220 CUUGACUAACACCACCACCAA 1798 UUGGUGGUGGUGUUAGUCAAGGG 2122  5369 AD-1334221 CUGUCAACCGAAGUGUGAGUA 1799 UACUCACACUUCGGUUGACAGCG 2123  5405 AD-1334222 AGAGUGGUUUGACGUGGACUA 1800 UAGUCCACGUCAAACCACUCUGU 2124  5429 AD-1334223 GGAAACUUUUGAAAACAUCAA 1801 UUGAUGUUUUCAAAAGUUUCCAU 2125  5480 AD-1334224 GCACCAAAGAGCAUAGAGUGA 1802 UCACUCUAUGCUCUUUGGUGCCC 2126  5526 AD-1334225 CGAGGUAAGCAUCGACCAGGA 1803 UCCUGGUCGAUGCUUACCUCGGG 2127  5564 AD-1334226 CUGACCUGCAGCCUGGAGACA 1804 UGUCUCCAGGCUGCAGGUCAGCA 2128  5595 AD-1334227 CUGCAAGAACGAAGACCAGAA 1805 UUCUGGUCUUCGUUCUUGCAGGU 2129  5624 AD-1334228 UGCUUCAACUACAACGUGCGA 1806 UCGCACGUUGUAGUUGAAGCACA 2130  5661 AD-1334229 UUGCUGUGACGACUACAGCCA 1807 UGGCUGUAGUCGUCACAGCAAAG 2131  5687 AD-1334230 GACGACCUGGAUCCUCACAAA 1808 UUUGUGAGGAUCCAGGUCGUCCC 2132  5762, 11120 AD-1334231 CGACCACAACAGCCACUACGA 1809 UCGUAGUGGCUGUUGUGGUCGGC 2133  5785,  7372, 11143, 12814, 13327 AD-1334232 UCCACCCUGAGAACAGCUCCA 1810 UGGAGCUGUUCUCAGGGUGGAGG 2134  5838 AD-1334233 UCCCAAAGUGCUGACCACCAA 1811 UUGGUGGUCAGCACUUUGGGAGG 2135  5861 AD-1334234 CAGCUCCAAAGCCACUCCCUA 1812 UAGGGAGUGGCUUUGGAGCUGGU 2136  5903,  7574, 11345, 13529 AD-1334235 CCAGUCCAGGGACUGCAACCA 1813 UGGUUGCAGUCCCUGGACUGGAG 2137  5926,  7597,  9697, 11368 AD-1334236 AGCACUGAGAAGCACAGCCAA 1814 UUGGCUGUGCUUCUCAGUGCUGG 2138  5954,  7625,  9725, 11396, 13580 AD-1334237 CUACCAGCGUUACACCCAUCA 1815 UGAUGGGUGUAACGCUGGUAGCU 2139  5986, 13015 AD-1334238 UUCCUCCCUGGGCACCACCUA 1816 UAGGUGGUGCCCAGGGAGGAAGA 2140  6011,  7682, 11453, 13637 AD-1334239 CCUAUCACAGACCACCACACA 1817 UGUGUGGUGGUCUGUGAUAGGCG 2141  6038,  7709,  9809, 11480, 13664 AD-1334240 CCACCAUGUCCACAGCCACAA 1818 UUGUGGCUGUGGACAUGGUGGCC 2142  6064,  7735,  9835, 11506, 13690 AD-1334241 UCCUCCACUCCAGAGACUGCA 1819 UGCAGUCUCUGGAGUGGAGGAGG 2143  6087, 11529 AD-1334242 CUCCACAGUGCUUACCGCCAA 1820 UUGGCGGUAAGCACUGUGGAGGU 2144  6113, 13739 AD-1334243 CAGGAACAGCUCACACUACCA 1821 UGGUAGUGUGAGCUGUUCCUGGG 2145  6184,  7855,  9955, 11626, 13810 AD-1334244 UGCCAACUACCACAACCACGA 1822 UCGUGGUUGUGGUAGUUGGCACU 2146  6208 AD-1334245 UCCAGUGUGGAUCAGCACAAA 1823 UUUGUGCUGAUCCACACUGGAGG 2147  6275,  7946, 10046, 11717, 13901 AD-1334246 ACCCACAACCAGAGGCUCCAA 1824 UUGGAGCCUCUGGUUGUGGGUGU 2148  6302, 10073 AD-1334247 CGCCACAGUGCUGACCACCAA 1825 UUGGUGGUCAGCACUGUGGCGGU 2149  6359,  8030, 10130, 11801, 14423 AD-1334248 GCCACUGGUUCUAUGGCAACA 1826 UGUUGCCAUAGAACCAGUGGCCA 2150  6393,  8064, 10164, 11835, 14034 AD-1334249 CUCCUCUAGCACACAGACCAA 1827 UUGGUCUGUGUGCUAGAGGAGGG 2151  6416,  8087, 10187, 11858, 14057 AD-1334250 GGCCACUACGAUCACGGCCAA 1828 UUGGCCGUGAUCGUAGUGGCCGU 2152  6464,  8135, 11906, 14105 AD-1334251 CUCCUCAACUCCUGGGACAAA 1829 UUUGUCCCAGGAGUUGAGGAGGG 2153  6503 AD-1334252 CAGCAACACAGUGACUCCCUA 1830 UAGGGAGUCACUGUGUUGCUGGU 2154  6572 AD-1334253 UGCCCUAGGGACCACCCACAA 1831 UUGUGGGUGGUCCCUAGGGCAGA 2155  6596,  8267, 10367, 12038 AD-1334254 AGUGCCGAACACCAUGGCCAA 1832 UUGGCCAUGGUGUUCGGCACUGG 2156  6623 AD-1334255 AGCCUGGACUUCGGCCACCUA 1833 UAGGUGGCCGAAGUCCAGGCUGU 2157  6692,  8363, 10463, 12134 AD-1334256 ACCCACAUCACAGAGCCUUCA 1834 UGAAGGCUCUGUGAUGUGGGUGG 2158  6729,  8400, 10500, 12171 AD-1334257 GGUGACUUCCCACACCCUAGA 1835 UCUAGGGUGUGGGAAGUCACCGU 2159  6752 AD-1334258 CAACCACCGGUACCACCCAGA 1836 UCUGGGUGGUACCGGUGGUUGCU 2160  6775,  8446, 12217 AD-1334259 CGACUCCAGCCCUUUCCAGCA 1837 UGCUGGAAAGGGCUGGAGUCGAG 2161  6799 AD-1334260 UAGCAGCAGAACCACCGAGUA 1838 UACUCGGUGGUUCUGCUGCUAGG 2162  6827 AD-1334261 ACCCAGCAAGACCCGCACCUA 1839 UAGGUGCGGGUCUUGCUGGGUGU 2163  6917,  8588, 10688, 12359 AD-1334262 CGGUGGUGACCAUGGGCUGUA 1840 UACAGCCCAUGGUCACCACCGUG 2164  6979,  8650 AD-1334263 GUGGCUGGACUACAGCUACCA 1841 UGGUAGCUGUAGUCCAGCCACUC 2165  7022,  8693, 10793, 12464 AD-1334264 UUGACACCUACUCCAACAUCA 1842 UGAUGUUGGAGUAGGUGUCAAAG 2166  7069,  8740, 10840, 12511 AD-1334265 UUGGGCCAGGUCGUGGAAUGA 1843 UCAUUCCACGACCUGGCCCAACU 2167  7173,  8844, 10944, 12615 AD-1334266 CCUGGACUUUGGCCUGGUCUA 1844 UAGACCAGGCCAAAGUCCAGGCU 2168  7196,  8867, 10967, 12638 AD-1334267 AUGUGCUUCAACUAUGAAAUA 1845 UAUUUCAUAGUUGAAGCACAUCU 2169  7248,  8919, 11019, 12690 AD-1334268 UGUGUUCUGCUGCAACUACGA 1846 UCGUAGUUGCAGCAGAACACACG 2170  7271,  8942, 11042, 12713 AD-1334269 CAGCUCUACGGCCAUGCCCUA 1847 UAGGGCAUGGCCGUAGAGCUGGU 2171  7316, 12758 AD-1334270 AUCCUCACAGAGCUGACCACA 1848 UGUGGUCAGCUCUGUGAGGAUCC 2172  7359,  9456, 12801, 13227, 13314 AD-1334271 CCACUACGACUGAGUCCACUA 1849 UAGUGGACUCAGUCGUAGUGGCU 2173  7384, 11155 AD-1334272 GACCACCUGGAUCCUCACAGA 1850 UCUGUGAGGAUCCAGGUGGUCCC 2174  7436,  9533, 11207, 13304, 13391, 14585 AD-1334273 GAGCACUACAGCCACCGUGAA 1851 UUCACGGUGGCUGUAGUGCUCGG 2175  7460,  9557, 11231, 13415 AD-1334274 CCUCCACCCAGGCAACUGCUA 1852 UAGCAGUUGCCUGGGUGGAGGAG 2176  7510, 11281, 13465 AD-1334275 GGCCACGACACCCACAGUCAA 1853 UUGACUGUGGGUGUCGUGGCCGU 2177  7553, 11324, 13508 AD-1334276 GCUCCAAAGCCACUCCCUUCA 1854 UGAAGGGAGUGGCUUUGGAGCUG 2178  7576, 11347 AD-1334277 ACAGCUACCAGCUUUACAGCA 1855 UGCUGUAAAGCUGGUAGCUGUGG 2179  7653, 11424, 13608 AD-1334278 UCCUCCACUCCAGAGACUGUA 1856 UACAGUCUCUGGAGUGGAGGAGG 2180  7758, 13713 AD-1334279 CCACAGUGCUUACCACCACGA 1857 UCGUGGUGGUAAGCACUGUGGAG 2181  7786,  9886, 11557 AD-1334280 GCUCACACUACCAAAGUGCUA 1858 UAGCACUUUGGUAGUGUGAGCUG 2182  7863 AD-1334281 UACCACAACCACGGGCUUCAA 1859 UUGAAGCCCGUGGUUGUGGUAGU 2183  6215,  7886,  9986, 11657, 13841 AD-1334282 CACGCUUCCAGUGUGGAUCAA 1860 UUGAUCCACACUGGAAGCGUGCG 2184  7940 AD-1334283 ACCCACAACCAGAGGUUCCAA 1861 UUGGAACCUCUGGUUGUGGGUGU 2185  7973 AD-1334284 UGACCACCACCACCACAACUA 1862 UAGUUGUGGUGGUGGUGGUCAGC 2186  6370,  8041, 10141, 11812, 14011 AD-1334285 CUCCUCAACUCCAGGGACAAA 1863 UUUGUCCCUGGAGUUGAGGAGGG 2187  8174, 10274, 11945, 14144 AD-1334286 CAGCAGCACAGUGACUCCCUA 1864 UAGGGAGUCACUGUGCUGCUGGU 2188  8243, 10343, 12014 AD-1334287 CACACACGGGCGAUCCCUGUA 1865 UACAGGGAUCGCCCGUGUGUGGU 2189  8315, 12086 AD-1334288 CCACCCAGCACUCGACUCCAA 1866 UUGGAGUCGAGUGCUGGGUGGUA 2190  6787,  8458, 10558, 12229 AD-1334289 CAGCCCUCACCCUAGCAGCAA 1867 UUGCUGCUAGGGUGAGGGCUGGA 2191  6815,  8486, 10586, 12257 AD-1334290 CUGGAUCCUCACAGAGCAGAA 1868 UUCUGCUCUGUGAGGAUCCAGGU 2192  9026 AD-1334291 CAGCCACUACGACCGCAACCA 1869 UGGUUGCGGUCGUAGUGGCUGCU 2193  9052 AD-1334292 UCCCAAAGUGCUGACCAGCAA 1870 UUGCUGGUCAGCACUUUGGGAGG 2194  9119 AD-1334293 CAGUUCCAAAGCCACUUCCUA 1871 UAGGAAGUGGCUUUGGAACUGGU 2195  9161, 12932 AD-1334294 CAAGGACUGCAACCACCCUUA 1872 UAAGGGUGGUUGCAGUCCUUGGA 2196  9190, 12961, 14242 AD-1334295 GUGCUGACAAGCACAGCCACA 1873 UGUGGCUGUGCUUGUCAGCACUG 2197  9213, 12984, 14265 AD-1334296 CACAGCUACCAGCUUUACACA 1874 UGUGUAAAGCUGGUAGCUGUGGA 2198  9239, 14291 AD-1334297 CUCCUUCACCCUUGGGACCAA 1875 UUGGUCCCAAGGGUGAAGGAGGG 2199  9266 AD-1334298 CCCAGAACAGACCACCACACA 1876 UGUGUGGUGGUCUGUUCUGGGAG 2200  9296, 13067 AD-1334299 CACCAUGUCCACAAUCCACCA 1877 UGGUGGAUUGUGGACAUGGUGGC 2201  9323, 13094 AD-1334300 CUCCACAGUGCUGACCACGAA 1878 UUCGUGGUCAGCACUGUGGAGGU 2202  9371, 13142 AD-1334301 GGCCACCAGUUCCAUGUCCAA 1879 UUGGACAUGGAACUGGUGGCCCU 2203  9407 AD-1334302 CCACUACAACUGCAGCCACUA 1880 UAGUGGCUGCAGUUGUAGUGGCU 2204  9481, 13252, 14533 AD-1334303 CAGCACUACAGCCACCGUGAA 1881 UUCACGGUGGCUGUAGUGCUGGG 2205  7460,  9557, 11231, 13415 AD-1334304 ACCCUCAAAGUGCUGACCAGA 1882 UCUGGUCAGCACUUUGAGGGUGC 2206  9630 AD-1334305 ACCCACAGUCAUCAGCUCCAA 1883 UUGGAGCUGAUGACUGUGGGUGU 2207  9662 AD-1334306 CCACUCCCUCCUCCAGUCCAA 1884 UUGGACUGGAGGAGGGAGUGGCU 2208  5914,  9685, 13540 AD-1334307 CUACCAGCGUUACAGCCAUCA 1885 UGAUGGCUGUAACGCUGGUAGCU 2209  9757 AD-1334308 CCUCUACUCCAGAGACUGUCA 1886 UGACAGUCUCUGGAGUAGAGGAG 2210  9859 AD-1334309 AGCUCACACUACCAAAGUGCA 1887 UGCACUUUGGUAGUGUGAGCUGU 2211  6191,  7862,  9962, 11633, 13817 AD-1334310 CUACCACAACCACGGGCUUCA 1888 UGAAGCCCGUGGUUGUGGUAGUC 2212  6214,  7885,  9985, 11656, 13840 AD-1334311 GGCCACUACGAUCACAGCCAA 1889 UUGGCUGUGAUCGUAGUGGCCGU 2213 10235 AD-1334312 CACCACGGCCACCACACACGA 1890 UCGUGUGUGGUGGCCGUGGUGUU 2214  8303, 10403, 12074 AD-1334313 CAGCAGCAACCACCAGUACCA 1891 UGGUACUGGUGGUUGCUGCUGGG 2215 10540 AD-1334314 CCUCCAGGACCACAGCCACAA 1892 UUGUGGCUGUGGUCCUGGAGGUG 2216 10663 AD-1334315 ACCACGGUGGUGACCACGGGA 1893 UCCCGUGGUCACCACCGUGGUUA 2217 10746, 12417 AD-1334316 CGGCCACGCCCUCCUCAACUA 1894 UAGUUGAGGAGGGCGUGGCCGUA 2218  5737, 11095 AD-1334317 CUGGAUCCUCACAAAGCUGAA 1895 UUCAGCUUUGUGAGGAUCCAGGU 2219 11126 AD-1334318 ACCCACAACCAGUGGCUCCAA 1896 UUGGAGCCACUGGUUGUGGGUGU 2220 11744, 13943 AD-1334319 GACGACCUGGAUCCUCACAGA 1897 UCUGUGAGGAUCCAGGUCGUCCC 2221  7349,  9020,  9446, 12791, 13217 AD-1334320 UGACCACAACAGCCACUACGA 1898 UCGUAGUGGCUGUUGUGGUCAGC 2222  5785,  7372, 11143, 12814, 13327 AD-1334321 AUCCACUGGAUCCACGGCCAA 1899 UUGGCCGUGGAUCCAGUGGAUGC 2223  5810,  7397,  9068, 11168, 12839, 13352, 14546 AD-1334322 CUCCCAAAGUGCUGACCAGCA 1900 UGCUGGUCAGCACUUUGGGAGGG 2224  9118, 12889 AD-1334323 CCACCAAAUCCACAGCUACCA 1901 UGGUAGCUGUGGAUUUGGUGGCU 2225  9229, 13000 AD-1334324 GGCCACCAGUUCCACGUCCAA 1902 UUGGACGUGGAACUGGUGGCCCU 2226 13178 AD-1334325 UGAGCACCACGGCCACGACAA 1903 UUGUCGUGGCCGUGGUGCUCACA 2227  7543, 11314, 13498 AD-1334326 CCAGGGACUGCAACUGCCCUA 1904 UAGGGCAGUUGCAGUCCCUGGAC 2228 13557 AD-1334327 CGCCAGAGUGCUGACCACCAA 1905 UUGGUGGUCAGCACUCUGGCGGU 2229 14000 AD-1334328 CAGGGACAACACCCAUCACCA 1906 UGGUGAUGGGUGUUGUCCCUGGA 2230 14155 AD-1334329 CUCCAAAGCCACUUCCUCCUA 1907 UAGGAGGAAGUGGCUUUGGAGCU 2231  9164, 12935, 14216 AD-1334330 UGCUGACAAGCACAGCCACAA 1908 UUGUGGCUGUGCUUGUCAGCACU 2232 14266 AD-1334331 CUCCACCCUGUGGACCACGUA 1909 UACGUGGUCCACAGGGUGGAGGA 2233 14321 AD-1334332 CCCAGCACAGACCACCACACA 1910 UGUGUGGUGGUCUGUGCUGGGAC 2234 14348 AD-1334333 GUCCACCAUGUCCACAAUCCA 1911 UGGAUUGUGGACAUGGUGGACAU 2235  9320, 13091, 14372 AD-1334334 CUCCUCUACUCCAGAGACCAA 1912 UUGGUCUCUGGAGUAGAGGAGGU 2236 14396 AD-1334335 CUCCACAGUGCUGACCACCAA 1913 UUGGUGGUCAGCACUGUGGAGGU 2237  6359,  8030, 10130, 11801, 14423 AD-1334336 GGCCACCAAUUCCACGGCCAA 1914 UUGGCCGUGGAAUUGGUGGCCCU 2238 14459 AD-1334337 UGACCACAACAGCCACUACAA 1915 UUGUAGUGGCUGUUGUGGUCAGC 2239 14521 AD-1334338 UGGAUCCACGGCCACCCUGUA 1916 UACAGGGUGGCCGUGGAUCCAGU 2240 14552 AD-1334339 GAGCACUAUAGCCACCGUGAA 1917 UUCACGGUGGCUAUAGUGCUCGG 2241 14609 AD-1334340 CCACUCUGGGAACAGCUCACA 1918 UGUGAGCUGUUCCCAGAGUGGAG 2242 14662 AD-1334341 CAUGGCCACUAUGCCCACAGA 1919 UCUGUGGGCAUAGUGGCCAUGGU 2243 14702 AD-1334342 UGCCUCCACGGUUCCCAGCUA 1920 UAGCUGGGAACCGUGGAGGCAGU 2244 14726 AD-1334343 GCCAACCUUCAGCGUGUCCAA 1921 UUGGACACGCUGAAGGUUGGCAG 2245 14795 AD-1334344 UCCUCCUCAGUCCUCACCACA 1922 UGUGGUGAGGACUGAGGAGGACA 2246 14820 AD-1334345 UCCCACUUCUCUACUCCCUGA 1923 UCAGGGAGUAGAGAAGUGGGAGC 2247 14865 AD-1334346 GCAUUUGGACAGUUUUUCUCA 1924 UGAGAAAAACUGUCCAAAUGCCC 2248 14895 AD-1334347 GAAGUCAUCUACAAUAAGACA 1925 UGUCUUAUUGUAGAUGACUUCCC 2249 14922 AD-1334348 CUGCCAUUUCUACGCAGUGUA 1926 UACACUGCGUAGAAAUGGCAGCC 2250 14954 AD-1334349 CACUGUGACAUUGACCGCUUA 1927 UAAGCGGUCAAUGUCACAGUGCU 2251 14982 AD-1334350 UGUGACAAUGCCAUCCCUCUA 1928 UAGAGGGAUGGCAUUGUCACAGC 2252 15075 AD-1334351 ACCCUGGAGAACUGCACGGUA 1929 UACCGUGCAGUUCUCCAGGGUCC 2253 15117 AD-1334352 GUGGGUGACAACCGUGUCGUA 1930 UACGACACGGUUGUCACCCACGC 2254 15147 AD-1334353 GACCCAAAGCCUGUGGCCAAA 1931 UUUGGCCACAGGCUUUGGGUCCA 2255 15174 AD-1334354 CUGCGUGAACAAGCACCUGCA 1932 UGCAGGUGCUUGUUCACGCAGGU 2256 15200 AD-1334355 UCAAAGUGUCGGACCCGAGCA 1933 UGCUCGGGUCCGACACUUUGAUG 2257 15223 AD-1334356 CCUGUGACUUCCACUAUGAGA 1934 UCUCAUAGUGGAAGUCACAGGGC 2258 15247 AD-1334357 GAGUGCAUCUGCAGCAUGUGA 1935 UCACAUGCUGCAGAUGCACUCGC 2259 15270 AD-1334358 CCCACUAUUCCACCUUUGACA 1936 UGUCAAAGGUGGAAUAGUGGGAG 2260 15298 AD-1334359 ACCUAUGUCCUCAUGAGAGAA 1937 UUCUCUCAUGAGGACAUAGGUGC 2261 15348 AD-1334360 CACGCUUUGGGAAUCUCAGCA 1938 UGCUGAGAUUCCCAAAGCGUGCA 2262 15376 AD-1334361 CUGGACAACCACUACUGCACA 1939 UGUGCAGUAGUGGUUGUCCAGGU 2263 15402 AD-1334362 CUCAGCAUCCACUACAAGUCA 1940 UGACUUGUAGUGGAUGCUGAGGG 2264 15462 AD-1334363 GUCCUCACUGUCACCAUGGUA 1941 UACCAUGGUGACAGUGAGGACGA 2265 15492 AD-1334364 CCUGAUCCUGUUUGACCAAAA 1942 UUUUGGUCAAACAGGAUCAGGCC 2266 15530 AD-1334365 AGCGGUUUCAGCAAGAACGGA 1943 UCCGUUCUUGCUGAAACCGCUGC 2267 15561 AD-1334366 CGUGUGGACAUUCCUGCCCUA 1944 UAGGGCAGGAAUGUCCACACGCA 2268 15615 AD-1334367 GUGAGCGUCACCUUCAAUGGA 1945 UCCAUUGAAGGUGACGCUCACGC 2269 15639 AD-1334368 AGCCUCUUCCACAACAACACA 1946 UGUGUUGUUGUGGAAGAGGCUGU 2270 15687 AD-1334369 UGCACCAACAACCAGAGGGAA 1947 UUCCCUCUGGUUGUUGGUGCAGG 2271 15726 AD-1334370 UGUCUCCAGCGGGACGGAACA 1948 UGUUCCGUCCCGCUGGAGACAGU 2272 15750 AD-1334371 CGCCAGUUGCAAGGACAUGGA 1949 UCCAUGUCCUUGCAACUGGCGGC 2273 15776 AD-1334372 CGACAGCAGAAAGGAUGGCUA 1950 UAGCCAUCCUUUCUGCUGUCGGG 2274 15815 AD-1334373 CCGCUCUGUGAUCUGAUGCUA 1951 UAGCAUCAGAUCACAGAGCGGCU 2275 15921 AD-1334374 CAGGUCUUUGCUGAGUGCCAA 1952 UUGGCACUCAGCAAAGACCUGGC 2276 15945 AD-1334375 GGGCCCAUUCUUCAACGCCUA 1953 UAGGCGUUGAAGAAUGGGCCCGG 2277 15980 AD-1334376 GAGGCUUACGCAGAGCUCUGA 1954 UCAGAGCUCUGCGUAAGCCUCCA 2278 16050 AD-1334377 AGUGUGCAGUGACUGGCGAGA 1955 UCUCGCCAGUCACUGCACACUCC 2279 16082 AD-1334378 CCACCAAAGUGUACAAGCCAA 1956 UUGGCUUGUACACUUUGGUGGGU 2280 16138 AD-1334379 CUGCAACUCUAGGAACCAGAA 1957 UUCUGGUUCCUAGAGUUGCAGGU 2281 16181 AD-1334380 CAGAUCCUCUUCAACGCACAA 1958 UUGUGCGUUGAAGAGGAUCUGGU 2282 16248 AD-1334381 GGGCAUCUGCGUGCAGGCCUA 1959 UAGGCCUGCACGCAGAUGCCCAU 2283 16271 AD-1334382 CGAUGGGUUUCCUAAAUUUCA 1960 UGAAAUUUAGGAAACCCAUCGGG 2284 16307 AD-1334383 GGUCAGCAACUGCCAGUCCUA 1961 UAGGACUGGCAGUUGCUGACCCA 2285 16340 AD-1334384 GAGGGUUCAGUGUCGGUGCAA 1962 UUGCACCGACACUGAACCCUCGU 2286 16371 AD-1334385 CGGCUUCGUAACCGUGACCAA 1963 UUGGUCACGGUUACGAAGCCGGG 2287 16445 AD-1334386 CGUGUGCAACACAACCACCUA 1964 UAGGUGGUUGUGUUGCACACGCA 2288 16505 AD-1334387 CAGGAGUCCAUCUGCACCCAA 1965 UUGGGUGCAGAUGGACUCCUGCC 2289 16557 AD-1334388 CUGUCCCACCUUCCGCUGCAA 1966 UUGCAGCGGAAGGUGGGACAGCA 2290 16592 AD-1334389 UCAGCUGUGUUCGUACAAUGA 1967 UCAUUGUACGAACACAGCUGAGG 2291 16616 AD-1334390 UUGGUGCAACCUUCCCAGGCA 1968 UGCCUGGGAAGGUUGCACCAACC 2292 16651 AD-1334391 UCCCUGCCACAUGUGUACCUA 1969 UAGGUACACAUGUGGCAGGGAAG 2293 16676 AD-1334392 ACGGUGCAAUGUCAGGAGGAA 1970 UUCCUCCUGACAUUGCACCGUUG 2294 16722 AD-1334393 CUGCAACAAUACUACCUGUCA 1971 UGACAGGUAGUAUUGUUGCAGGC 2295 16745 AD-1334394 GGGCUUUGAGUACAAGAGAGA 1972 UCUCUCUUGUACUCAAAGCCCUG 2296 16769 AD-1334395 GUCCAGCUGAAUGAAACCUGA 1973 UCAGGUUUCAUUCAGCUGGACUG 2297 16851 AD-1334396 CAACAGCCAUGUGGACAACUA 1974 UAGUUGUCCACAUGGCUGUUGAC 2298 16874 AD-1334397 CCGUGUACCUCUGUGAGGCUA 1975 UAGCCUCACAGAGGUACACGGUG 2299 16897 AD-1334398 GGUGGAGUCCAUUUGCUGACA 1976 UGUCAGCAAAUGGACUCCACCCU 2300 16920 AD-1334399 CUGCCCAGAUGUGUCCAGCUA 1977 UAGCUGGACACAUCUGGGCAGGA 2301 16955 AD-1334400 GCUGCUACUCCUGUGAGGAGA 1978 UCUCCUCACAGGAGUAGCAGCAG 2302 17002 AD-1334401 UCCUGUCAAGUCCGCAUCAAA 1979 UUUGAUGCGGACUUGACAGGAGU 2303 17025 AD-1334402 GACCAUCCUGUGGCACCAGGA 1980 UCCUGGUGCCACAGGAUGGUCGU 2304 17048 AD-1334403 GGUCAACAUCACCUUCUGCGA 1981 UCGCAGAAGGUGAUGUUGACCUC 2305 17081 AD-1334404 CGUCCAAGUACUCAGCAGAGA 1982 UCUCUGCUGAGUACUUGGACGCU 2306 17119 AD-1334405 AUGCAGCACCAGUGCACCUGA 1983 UCAGGUGCACUGGUGCUGCAUGG 2307 17148 AD-1334406 GCCCUUGCACUGUCCUAACGA 1984 UCGUUAGGACAGUGCAAGGGCAC 2308 17201 AD-1334407 CUGCACACCUACACCCACGUA 1985 UACGUGGGUGUAGGUGUGCAGGA 2309 17232 AD-1334408 GCACGCCCUUCUGUGUCCCUA 1986 UAGGGACACAGAAGGGCGUGCAG 2310 17266 AD-1334409 ACUGCUGUCUGAGAACGUUCA 1987 UGAACGUUCUCAGACAGCAGUGG 2311 17334 AD-1334410 CAUGCUCUGUCCACCUGGAGA 1988 UCUCCAGGUGGACAGAGCAUGGG 2312 17366 AD-1334411 GCAUUGUCUGAUCAUGAAAAA 1989 UUUUUCAUGAUCAGACAAUGCAC 2313 17395 AD-1334412 GGCGCCACUCAGGAGUCCUAA 1990 UUAGGACUCCUGAGUGGCGCCCU 2314 17542 AD-1334413 CUCCCUGAUGUCACUGGGACA 1991 UGUCCCAGUGACAUCAGGGAGGG 2315 17598 AD-1334414 CUGGAACAAACUAAGCAUGUA 1992 UACAUGCUUAGUUUGUUCCAGGG 2316 17621 AD-1334415 GCACGGAUUCCAGCUGGCCAA 1993 UUGGCCAGCUGGAAUCCGUGCUG 2317 17682 AD-1334416 GACAGGCUGGUCCAGGCAAGA 1994 UCUUGCCUGGACCAGCCUGUCUG 2318 17720 AD-1334417 GCUGCCAGGAAGCUGCGACAA 1995 UUGUCGCAGCUUCCUGGCAGCAG 2319 17747 AD-1334418 GCAGGGUAACUCAGGGCUGAA 1996 UUCAGCCCUGAGUUACCCUGCAG 2320 17796 AD-1334419 GCAACGGCCAGGUCAGAGAGA 1997 UCUCUCUGACCUGGCCGUUGCGA 2321 17820 AD-1334420 AGCCCAGUUUUGCAAAUAAAA 1998 UUUUAUUUGCAAAACUGGGCUGG 2322 17874

TABLE 5 Modified Sense and Antisense Strand MUC5B dsRNA Sequences SEQ SEQ ID ID Duplex Name Sense Sequence 5′ to 3′ NO: Antisense Sequence NO: AD-1334097 ususggcuCfuGfGfCfggccaugcsusa 2323 VPusAfsgcaUfgGfCfcgccAfgAfgccaascsa 2647 AD-1334098 csusgggaGfaAfUfGfcagggcacsasa 2324 VPusUfsgugCfcCfUfgcauUfcUfcccagscsu 2648 AD-1334099 gscsgcguGfaGfCfUfuuguuccascsa 2325 VPusGfsuggAfaCfAfaagcUfcAfcgcgcscsg 2649 AD-1334100 usgsggcgGfgUfGfUfgcagcaccsusa 2326 VPusAfsgguGfcUfGfcacaCfcCfgcccasusu 2650 AD-1334101 cscsacuaCfaAfGfAfccuucgacsgsa 2327 VPusCfsgucGfaAfGfgucuUfgUfaguggsasa 2651 AD-1334102 cscsuuugCfaAfCfUfacguguucsusa 2328 VPusAfsgaaCfaCfGfuaguUfgCfaaaggscsc 2652 AD-1334103 gsasggacUfuCfAfAfcguccagcsusa 2329 VPusAfsgcuGfgAfCfguugAfaGfuccucsgsu 2653 AD-1334104 ascsccguGfuUfGfUfcaucaaggscsa 2330 VPusGfsccuUfgAfUfgacaAfcAfcgggusgsa 2654 AD-1334105 gsgscuccGfuCfCfUfcaucaaugsgsa 2331 VPusCfscauUfgAfUfgaggAfcGfgagccsgsu 2655 AD-1334106 gscsugccUfuAfCfAfgccgcacusgsa 2332 VPusCfsaguGfcGfGfcuguAfaGfgcagcsusc 2656 AD-1334107 gsascuacAfuCfAfAfggucagcasusa 2333 VPusAfsugcUfgAfCfcuugAfuGfuagucscsc 2657 AD-1334108 gscsugacAfuUfCfCfuguggaacsgsa 2334 VPusCfsguuCfcAfCfaggaAfuGfucagcsasc 2658 AD-1334109 csusggauCfcCfAfAfauacgccasasa 2335 VPusUfsuggCfgUfAfuuugGfgAfuccagscsu 2659 AD-1334110 gscscuucAfaCfGfAfguucuaugscsa 2336 VPusGfscauAfgAfAfcucgUfuGfaaggcscsg 2660 AD-1334111 asasccugCfaGfAfAfguuggaugsgsa 2337 VPusCfscauCfcAfAfcuucUfgCfagguuscsc 2661 AD-1334112 usgscacgGfaCfGfAfggagggcasusa 2338 VPusAfsugcCfcUfCfcucgUfcCfgugcasgsu 2662 AD-1334113 ususugcgGfaGfUfGfccacgcacsusa 2339 VPusAfsgugCfgUfGfgcacUfcCfgcaaasgsg 2663 AD-1334114 gsascagcAfcUfGfCfguaccuggscsa 2340 VPusGfsccaGfgUfAfcgcaGfuGfcugucscsa 2664 AD-1334115 gscscaccUfuUfGfUfggaauacuscsa 2341 VPusGfsaguAfuUfCfcacaAfaGfguggcsasc 2665 AD-1334116 csusggagGfuGfCfCfcugagcucsusa 2342 VPusAfsgagCfuCfAfgggcAfcCfuccagsusu 2666 AD-1334117 csuscaacAfuGfCfAfgcaccaggsasa 2343 VPusUfsccuGfgUfGfcugcAfuGfuugagsgsg 2667 AD-1334118 ascsccugCfaCfGfGfacaccugcsusa 2344 VPusAfsgcaGfgUfGfuccgUfgCfagggusgsa 2668 AD-1334119 gsgsaccaCfuGfUfGfuggacggcsusa 2345 VPusAfsgccGfuCfCfacacAfgUfgguccsusc 2669 AD-1334120 gscsuggaUfgAfCfAfucacgcacsusa 2346 VPusAfsgugCfgUfGfauguCfaUfccagcsasc 2670 AD-1334121 csusccuuCfaAfCfAfccaccugcsasa 2347 VPusUfsgcaGfgUfGfguguUfgAfaggagsgsu 2671 AD-1334122 csusauggCfaGfUfGfccaggaccsusa 2348 VPusAfsgguCfcUfGfgcacUfgCfcauagscsc 2672 AD-1334123 ascscuauGfaUfGfAfgaaacucusasa 2349 VPusUfsagaGfuUfUfcucaUfcAfuaggusgsg 2673 AD-1334124 asgscuacGfuUfCfUfguccaagasasa 2350 VPusUfsucuUfgGfAfcagaAfcGfuagcusgsc 2674 AD-1334125 gsascagcAfgCfUfUfcaccgugcsusa 2351 VPusAfsgcaCfgGfUfgaagCfuGfcugucsgsg 2675 AD-1334126 gsgsacaaCfgAfGfAfacugccugsasa 2352 VPusUfscagGfcAfGfuucuCfgUfuguccsgsu 2676 AD-1334127 uscscucaAfcUfCfCfaucuacacsgsa 2353 VPusCfsgugUfaGfAfuggaGfuUfgaggasasc 2677 AD-1334128 gscscaacAfuCfAfCfccuguucascsa 2354 VPusGfsugaAfcAfGfggugAfuGfuuggcsusg 2678 AD-1334129 uscsgagcUfuCfUfUfcaucguggsusa 2355 VPusAfsccaCfgAfUfgaagAfaGfcucgasgsg 2679 AD-1334130 gscscacuCfaUfGfCfagguguuusgsa 2356 VPusCfsaaaCfaCfCfugcaUfgAfguggcsasc 2680 AD-1334131 gsgsgaacUfuCfAfAfccagaaccsasa 2357 VPusUfsgguUfcUfGfguugAfaGfuucccsasc 2681 AD-1334132 usgsacgaCfuUfCfAfcggcccucsasa 2358 VPusUfsgagGfgCfCfgugaAfgUfcgucasgsc 2682 AD-1334133 asgsccuuCfgCfCfAfacaccuggsasa 2359 VPusUfsccaGfgUfGfuuggCfgAfaggcusgsc 2683 AD-1334134 cscsaggaAfcAfGfCfuuugaggascsa 2360 VPusGfsuccUfcAfAfagcuGfuUfccuggscsa 2684 AD-1334135 gsusggagAfaUfGfAfgaacuacgscsa 2361 VPusGfscguAfgUfUfcucaUfuCfuccacsasc 2685 AD-1334136 csasacagUfgCfCfUfucucgcgcsusa 2362 VPusAfsgcgCfgAfGfaaggCfaCfuguugsgsg 2686 AD-1334137 csusuccaCfuCfGfAfacugcaugsusa 2363 VPusAfscauGfcAfGfuucgAfgUfggaagsgsg 2687 AD-1334138 csasccugCfaAfCfUfgugagcggsasa 2364 VPusUfsccgCfuCfAfcaguUfgCfaggugsusc 2688 AD-1334139 cscsuccuAfuGfUfGfcacgccugsusa 2365 VPusAfscagGfcGfUfgcacAfuAfggaggsasc 2689 AD-1334140 gsgscguaCfaGfCfUfcagcgacusgsa 2366 VPusCfsaguCfgCfUfgagcUfgUfacgccscsu 2690 AD-1334141 ascscaagUfaCfAfUfgcagaacusgsa 2367 VPusCfsaguUfcUfGfcaugUfaCfuuggusgsc 2691 AD-1334142 usascgccUfaCfGfUfgguggaugscsa 2368 VPusGfscauCfcAfCfcacgUfaGfgcguasgsc 2692 AD-1334143 gscsagcgUfuUfCfCfuucgugccsusa 2369 VPusAfsggcAfcGfAfaggaAfaCfgcugcsasg 2693 AD-1334144 gscsaccuUfcCfUfCfaaugacgcsgsa 2370 VPusCfsgcgUfcAfUfugagGfaAfggugcscsc 2694 AD-1334145 csusggagAfgGfUfGfgugcacgascsa 2371 VPusGfsucgUfgCfAfccacCfuCfuccagsgsa 2695 AD-1334146 cscsguguGfuUfCfAfuguacgggsusa 2372 VPusAfscccGfuAfCfaugaAfcAfcacggscsg 2696 AD-1334147 cscsucucUfgCfAfGfaaaagcacsasa 2373 VPusUfsgugCfuUfUfucugCfaGfagaggscsu 2697 AD-1334148 gsgsacugCfaGfCfAfacagcucgsgsa 2374 VPusCfscgaGfcUfGfuugcUfgCfaguccsasg 2698 AD-1334149 csusguuuCfaGfCfAfcacacugcsgsa 2375 VPusCfsgcaGfuGfUfgugcUfgAfaacagscsc 2699 AD-1334150 csusgcauUfgCfCfGfaggaggacsusa 2376 VPusAfsgucCfuCfCfucggCfaAfugcagscsc 2700 AD-1334151 csasccuaCfaAfGfCfcuggagagsasa 2377 VPusUfscucUfcCfAfggcuUfgUfaggugsgsc 2701 AD-1334152 csgsacugCfaAfCfAfccugcaccsusa 2378 VPusAfsgguGfcAfGfguguUfgCfagucgsasc 2702 AD-1334153 gsasaccgGfaGfGfUfgggagugcsasa 2379 VPusUfsgcaCfuCfCfcaccUfcCfgguucscsu 2703 AD-1334154 usgsgccaCfuUfCfAfucaccuuusgsa 2380 VPusCfsaaaGfgUfGfaugaAfgUfggccasusc 2704 AD-1334155 csgsaucgCfuAfCfAfgcuuugaasgsa 2381 VPusCfsuucAfaAfGfcuguAfgCfgaucgscsc 2705 AD-1334156 gscsugcgAfgUfAfCfaucuuggcscsa 2382 VPusGfsgccAfaGfAfuguaCfuCfgcagcsusg 2706 AD-1334157 csusuccgCfaUfCfGfucaccgagsasa 2383 VPusUfscucGfgUfGfacgaUfgCfggaagsgsu 2707 AD-1334158 gscscaucAfaGfCfUfcuucguggsasa 2384 VPusUfsccaCfgAfAfgagcUfuGfauggcscsu 2708 AD-1334159 usascgagCfuGfAfUfccuccaagsasa 2385 VPusUfscuuGfgAfGfgaucAfgCfucguasgsc 2709 AD-1334160 gsasccuuUfaAfGfGfcgguggcgsasa 2386 VPusUfscgcCfaCfCfgccuUfaAfaggucscsc 2710 AD-1334161 ascsccuaCfaAfGfAfuacgcuacsasa 2387 VPusUfsguaGfcGfUfaucuUfgUfagggusgsg 2711 AD-1334162 ususccugGfuCfAfUfcgagacccsasa 2388 VPusUfsgggUfcUfCfgaugAfcCfaggaasgsa 2712 AD-1334163 cscsagcgUfgUfUfCfauccgacusgsa 2389 VPusCfsaguCfgGfAfugaaCfaCfgcuggsusc 2713 AD-1334164 csasggacUfaCfAfAfgggcagggsusa 2390 VPusAfscccUfgCfCfcuugUfaGfuccugsgsu 2714 AD-1334165 gsgsgaacUfuCfGfAfcgacaaugscsa 2391 VPusGfscauUfgUfCfgucgAfaGfuucccsgsc 2715 AD-1334166 csasaugaCfuUfUfGfccacgcgusasa 2392 VPusUfsacgCfgUfGfgcaaAfgUfcauugsasu 2716 AD-1334167 gscsacugGfaGfUfUfugggaacasgsa 2393 VPusCfsuguUfcCfCfaaacUfcCfagugcsgsu 2717 AD-1334168 gsgscccaGfaAfGfCfagugcagcsasa 2394 VPusUfsgcuGfcAfCfugcuUfcUfgggccscsa 2718 AD-1334169 cscsagguUfgAfCfUfccaccaagsusa 2395 VPusAfscuuGfgUfGfgaguCfaAfccuggsgsa 2719 AD-1334170 csgsaggcCfuGfCfGfugaacgacsgsa 2396 VPusCfsgucGfuUfCfacgcAfgGfccucgsusa 2720 AD-1334171 ascsugcgAfgUfGfUfuucugcacsgsa 2397 VPusCfsgugCfaGfAfaacaCfuCfgcaguscsg 2721 AD-1334172 usgsugugUfgUfCfCfuggcggacsusa 2398 VPusAfsgucCfgCfCfaggaCfaCfacacasgsg 2722 AD-1334173 usgsuucuGfuGfAfCfuucuacaascsa 2399 VPusGfsuugUfaGfAfagucAfcAfgaacasasg 2723 AD-1334174 usgsugagUfgGfCfAfcuaccagcscsa 2400 VPusGfsgcuGfgUfAfgugcCfaCfucacasgsc 2724 AD-1334175 gscsugcuAfcCfCfGfaagugcccsasa 2401 VPusUfsgggCfaCfUfucggGfuAfgcagescsu 2725 AD-1334176 asgscccuUfcUfUfCfaaugaggascsa 2402 VPusGfsuccUfcAfUfugaaGfaAfgggcusgsg 2726 AD-1334177 asasgugcGfuGfGfCfccagugugsgsa 2403 VPusCfscacAfcUfGfggccAfcGfcacuuscsa 2727 AD-1334178 csusacgaCfaAfGfGfacggaaacsusa 2404 VPusAfsguuUfcCfGfuccuUfgUfcguagscsa 2728 AD-1334179 usgsacguCfgGfUfGfcaagggucscsa 2405 VPusGfsgacCfcUfUfgcacCfgAfcgucasusa 2729 AD-1334180 cscsagagCfuGfUfAfacugcacascsa 2406 VPusGfsuguGfcAfGfuuacAfgCfucuggscsa 2730 AD-1334181 csasgugcGfcUfCfAfcagccuugsasa 2407 VPusUfscaaGfgCfUfgugaGfcGfcacugsgsa 2731 AD-1334182 csusgcacCfuAfUfGfaggacaggsasa 2408 VPusUfsccuGfuCfCfucauAfgGfugcagsgsu 2732 AD-1334183 csasggacGfuCfAfUfcuacaacascsa 2409 VPusGfsuguUfgUfAfgaugAfcGfuccugsgsu 2733 AD-1334184 csgsccugCfuUfGfAfucgccaucsusa 2410 VPusAfsgauGfgCfGfaucaAfgCfaggcgscsc 2734 AD-1334185 ascscaucAfuCfAfGfgaaggcugsusa 2411 VPusAfscagCfcUfUfccugAfuGfauggusgsc 2735 AD-1334186 csascaacGfcCfAfUfucaccuucsasa 2412 VPusUfsgaaGfgUfGfaaugGfcGfuugugsgsc 2736 AD-1334187 uscscaccGfuGfUfGfuguccgcgsasa 2413 VPusUfscgcGfgAfCfacacAfcGfguggasgsa 2737 AD-1334188 uscscagcUfgGfUfAfcaaugggcsasa 2414 VPusUfsgccCfaUfUfguacCfaGfcuggascsc 2738 AD-1334189 csgsgagaCfuUfUfGfagacguuusgsa 2415 VPusCfsaaaCfgUfCfucaaAfgUfcuccgscsc 2739 AD-1334190 gsasggguAfcCfAfGfguaugcccsusa 2416 VPusAfsgggCfaUfAfccugGfuAfcccucsusc 2740 AD-1334191 csusggcuGfaCfAfUfcgagugccsgsa 2417 VPusCfsggcAfcUfCfgaugUfcAfgccagscsa 2741 AD-1334192 csusucccGfaCfAfUfgccgcuggsasa 2418 VPusUfsccaGfcGfGfcaugUfcGfggaagscsu 2742 AD-1334193 csasggugGfaCfUfGfugaccgcasusa 2419 VPusAfsugcGfgUfCfacagUfcCfaccugscsu 2743 AD-1334194 csgsccaaCfaGfCfCfaacagaguscsa 2420 VPusGfsacuCfuGfUfuggcUfgUfuggcgscsa 2744 AD-1334195 uscsugucAfcGfAfCfuacgagcusgsa 2421 VPusCfsagcUfcGfUfagucGfuGfacagasgsc 2745 AD-1334196 uscsucugCfuGfCfGfaauacgugscsa 2422 VPusGfscacGfuAfUfucgcAfgCfagagasasc 2746 AD-1334197 csascggaGfcCfUfGfcugugccusasa 2423 VPusUfsaggCfaCfAfgcagGfcUfccgugscsu 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2591 VPusCfscguUfcUfUfgcugAfaAfccgcusgsc 2915 AD-1334366 csgsugugGfaCfAfUfuccugcccsusa 2592 VPusAfsgggCfaGfGfaaugUfcCfacacgscsa 2916 AD-1334367 gsusgagcGfuCfAfCfcuucaaugsgsa 2593 VPusCfscauUfgAfAfggugAfcGfcucacsgsc 2917 AD-1334368 asgsccucUfuCfCfAfcaacaacascsa 2594 VPusGfsuguUfgUfUfguggAfaGfaggcusgsu 2918 AD-1334369 usgscaccAfaCfAfAfccagagggsasa 2595 VPusUfscccUfcUfGfguugUfuGfgugcasgsg 2919 AD-1334370 usgsucucCfaGfCfGfggacggaascsa 2596 VPusGfsuucCfgUfCfccgcUfgGfagacasgsu 2920 AD-1334371 csgsccagUfuGfCfAfaggacaugsgsa 2597 VPusCfscauGfuCfCfuugcAfaCfuggcgsgsc 2921 AD-1334372 csgsacagCfaGfAfAfaggauggcsusa 2598 VPusAfsgccAfuCfCfuuucUfgCfugucgsgsg 2922 AD-1334373 cscsgcucUfgUfGfAfucugaugcsusa 2599 VPusAfsgcaUfcAfGfaucaCfaGfageggscsu 2923 AD-1334374 csasggucUfuUfGfCfugagugccsasa 2600 VPusUfsggcAfcUfCfagcaAfaGfaccugsgsc 2924 AD-1334375 gsgsgcccAfuUfCfUfucaacgccsusa 2601 VPusAfsggcGfuUfGfaagaAfuGfggcccsgsg 2925 AD-1334376 gsasggcuUfaCfGfCfagagcucusgsa 2602 VPusCfsagaGfcUfCfugcgUfaAfgccucscsa 2926 AD-1334377 asgsugugCfaGfUfGfacuggcgasgsa 2603 VPusCfsucgCfcAfGfucacUfgCfacacuscsc 2927 AD-1334378 cscsaccaAfaGfUfGfuacaagccsasa 2604 VPusUfsggcUfuGfUfacacUfuUfgguggsgsu 2928 AD-1334379 csusgcaaCfuCfUfAfggaaccagsasa 2605 VPusUfscugGfuUfCfcuagAfgUfugcagsgsu 2929 AD-1334380 csasgaucCfuCfUfUfcaacgcacsasa 2606 VPusUfsgugCfgUfUfgaagAfgGfaucugsgsu 2930 AD-1334381 gsgsgcauCfuGfCfGfugcaggccsusa 2607 VPusAfsggcCfuGfCfacgcAfgAfugcccsasu 2931 AD-1334382 csgsauggGfuUfUfCfcuaaauuuscsa 2608 VPusGfsaaaUfuUfAfggaaAfcCfcaucgsgsg 2932 AD-1334383 gsgsucagCfaAfCfUfgccaguccsusa 2609 VPusAfsggaCfuGfGfcaguUfgCfugaccscsa 2933 AD-1334384 gsasggguUfcAfGfUfgucggugcsasa 2610 VPusUfsgcaCfcGfAfcacuGfaAfcccucsgsu 2934 AD-1334385 csgsgcuuCfgUfAfAfccgugaccsasa 2611 VPusUfsgguCfaCfGfguuaCfgAfagccgsgsg 2935 AD-1334386 csgsugugCfaAfCfAfcaaccaccsusa 2612 VPusAfsgguGfgUfUfguguUfgCfacacgscsa 2936 AD-1334387 csasggagUfcCfAfUfcugcacccsasa 2613 VPusUfsgggUfgCfAfgaugGfaCfuccugscsc 2937 AD-1334388 csusguccCfaCfCfUfuccgcugcsasa 2614 VPusUfsgcaGfcGfGfaaggUfgGfgacagscsa 2938 AD-1334389 uscsagcuGfuGfUfUfcguacaausgsa 2615 VPusCfsauuGfuAfCfgaacAfcAfgcugasgsg 2939 AD-1334390 ususggugCfaAfCfCfuucccaggscsa 2616 VPusGfsccuGfgGfAfagguUfgCfaccaascsc 2940 AD-1334391 uscsccugCfcAfCfAfuguguaccsusa 2617 VPusAfsgguAfcAfCfauguGfgCfagggasasg 2941 AD-1334392 ascsggugCfaAfUfGfucaggaggsasa 2618 VPusUfsccuCfcUfGfacauUfgCfaccgususg 2942 AD-1334393 csusgcaaCfaAfUfAfcuaccuguscsa 2619 VPusGfsacaGfgUfAfguauUfgUfugcagsgsc 2943 AD-1334394 gsgsgcuuUfgAfGfUfacaagagasgsa 2620 VPusCfsucuCfuUfGfuacuCfaAfagcccsusg 2944 AD-1334395 gsusccagCfuGfAfAfugaaaccusgsa 2621 VPusCfsaggUfuUfCfauucAfgCfuggacsusg 2945 AD-1334396 csasacagCfcAfUfGfuggacaacsusa 2622 VPusAfsguuGfuCfCfacauGfgCfuguugsasc 2946 AD-1334397 cscsguguAfcCfUfCfugugaggcsusa 2623 VPusAfsgccUfcAfCfagagGfuAfcacggsusg 2947 AD-1334398 gsgsuggaGfuCfCfAfuuugcugascsa 2624 VPusGfsucaGfcAfAfauggAfcUfccaccscsu 2948 AD-1334399 csusgcccAfgAfUfGfuguccagcsusa 2625 VPusAfsgcuGfgAfCfacauCfuGfggcagsgsa 2949 AD-1334400 gscsugcuAfcUfCfCfugugaggasgsa 2626 VPusCfsuccUfcAfCfaggaGfuAfgcagcsasg 2950 AD-1334401 uscscuguCfaAfGfUfccgcaucasasa 2627 VPusUfsugaUfgCfGfgacuUfgAfcaggasgsu 2951 AD-1334402 gsasccauCfcUfGfUfggcaccagsgsa 2628 VPusCfscugGfuGfCfcacaGfgAfuggucsgsu 2952 AD-1334403 gsgsucaaCfaUfCfAfccuucugcsgsa 2629 VPusCfsgcaGfaAfGfgugaUfgUfugaccsusc 2953 AD-1334404 csgsuccaAfgUfAfCfucagcagasgsa 2630 VPusCfsucuGfcUfGfaguaCfuUfggacgscsu 2954 AD-1334405 asusgcagCfaCfCfAfgugcaccusgsa 2631 VPusCfsaggUfgCfAfcuggUfgCfugcausgsg 2955 AD-1334406 gscsccuuGfcAfCfUfguccuaacsgsa 2632 VPusCfsguuAfgGfAfcaguGfcAfagggcsasc 2956 AD-1334407 csusgcacAfcCfUfAfcacccacgsusa 2633 VPusAfscguGfgGfUfguagGfuGfugcagsgsa 2957 AD-1334408 gscsacgcCfcUfUfCfugugucccsusa 2634 VPusAfsgggAfcAfCfagaaGfgGfcgugcsasg 2958 AD-1334409 ascsugcuGfuCfUfGfagaacguuscsa 2635 VPusGfsaacGfuUfCfucagAfcAfgcagusgsg 2959 AD-1334410 csasugcuCfuGfUfCfcaccuggasgsa 2636 VPusCfsuccAfgGfUfggacAfgAfgcaugsgsg 2960 AD-1334411 gscsauugUfcUfGfAfucaugaaasasa 2637 VPusUfsuuuCfaUfGfaucaGfaCfaaugcsasc 2961 AD-1334412 gsgscgccAfcUfCfAfggaguccusasa 2638 VPusUfsaggAfcUfCfcugaGfuGfgcgccscsu 2962 AD-1334413 csuscccuGfaUfGfUfcacugggascsa 2639 VPusGfsuccCfaGfUfgacaUfcAfgggagsgsg 2963 AD-1334414 csusggaaCfaAfAfCfuaagcaugsusa 2640 VPusAfscauGfcUfUfaguuUfgUfuccagsgsg 2964 AD-1334415 gscsacggAfuUfCfCfagcuggccsasa 2641 VPusUfsggcCfaGfCfuggaAfuCfcgugcsusg 2965 AD-1334416 gsascaggCfuGfGfUfccaggcaasgsa 2642 VPusCfsuugCfcUfGfgaccAfgCfcugucsusg 2966 AD-1334417 gscsugccAfgGfAfAfgcugcgacsasa 2643 VPusUfsgucGfcAfGfcuucCfuGfgcagcsasg 2967 AD-1334418 gscsagggUfaAfCfUfcagggcugsasa 2644 VPusUfscagCfcCfUfgaguUfaCfccugcsasg 2968 AD-1334419 gscsaacgGfcCfAfGfgucagagasgsa 2645 VPusCfsucuCfuGfAfccugGfcCfguugcsgsa 2969 AD-1334420 asgscccaGfuUfUfUfgcaaauaasasa 2646 VPusUfsuuaUfuUfGfcaaaAfcUfgggcusgsg 2970 SEQ mRNA Target Sequence ID Duplex Name 5′ to 3′ NO: AD-1334097 TGTTGGCTCTGGCGGCCATGCTC 2971 AD-1334098 AGCTGGGAGAATGCAGGGCACAC 2972 AD-1334099 CGGCGCGTGAGCTTTGTTCCACC 2973 AD-1334100 AATGGGCGGGTGTGCAGCACCTG 2974 AD-1334101 TTCCACTACAAGACCTTCGACGG 2975 AD-1334102 GGCCTTTGCAACTACGTGTTCTC 2976 AD-1334103 ACGAGGACTTCAACGTCCAGCTA 2977 AD-1334104 TCACCCGTGTTGTCATCAAGGCC 2978 AD-1334105 ACGGCTCCGTCCTCATCAATGGG 2979 AD-1334106 GAGCTGCCTTACAGCCGCACTGG 2980 AD-1334107 GGGACTACATCAAGGTCAGCATC 2981 AD-1334108 GTGCTGACATTCCTGTGGAACGG 2982 AD-1334109 AGCTGGATCCCAAATACGCCAAC 2983 AD-1334110 CGGCCTTCAACGAGTTCTATGCC 2984 AD-1334111 GGAACCTGCAGAAGTTGGATGGG 2985 AD-1334112 ACTGCACGGACGAGGAGGGCATC 2986 AD-1334113 CCTTTGCGGAGTGCCACGCACTG 2987 AD-1334114 TGGACAGCACTGCGTACCTGGCC 2988 AD-1334115 GTGCCACCTTTGTGGAATACTCA 2989 AD-1334116 AACTGGAGGTGCCCTGAGCTCTG 2990 AD-1334117 CCCTCAACATGCAGCACCAGGAG 2991 AD-1334118 TCACCCTGCACGGACACCTGCTC 2992 AD-1334119 GAGGACCACTGTGTGGACGGCTG 2993 AD-1334120 GTGCTGGATGACATCACGCACTC 2994 AD-1334121 ACCTCCTTCAACACCACCTGCAG 2995 AD-1334122 GGCTATGGCAGTGCCAGGACCTG 2996 AD-1334123 CCACCTATGATGAGAAACTCTAC 2997 AD-1334124 GCAGCTACGTTCTGTCCAAGAAA 2998 AD-1334125 CCGACAGCAGCTTCACCGTGCTG 2999 AD-1334126 ACGGACAACGAGAACTGCCTGAA 3000 AD-1334127 GTTCCTCAACTCCATCTACACGC 3001 AD-1334128 CAGCCAACATCACCCTGTTCACA 3002 AD-1334129 CCTCGAGCTTCTTCATCGTGGTG 3003 AD-1334130 GTGCCACTCATGCAGGTGTTTGT 3004 AD-1334131 GTGGGAACTTCAACCAGAACCAG 3005 AD-1334132 GCTGACGACTTCACGGCCCTCAG 3006 AD-1334133 GCAGCCTTCGCCAACACCTGGAA 3007 AD-1334134 TGCCAGGAACAGCTTTGAGGACC 3008 AD-1334135 GTGTGGAGAATGAGAACTACGCC 3009 AD-1334136 CCCAACAGTGCCTTCTCGCGCTG 3010 AD-1334137 CCCTTCCACTCGAACTGCATGTT 3011 AD-1334138 GACACCTGCAACTGTGAGCGGAG 3012 AD-1334139 GTCCTCCTATGTGCACGCCTGTG 3013 AD-1334140 AGGGCGTACAGCTCAGCGACTGG 3014 AD-1334141 GCACCAAGTACATGCAGAACTGC 3015 AD-1334142 GCTACGCCTACGTGGTGGATGCC 3016 AD-1334143 CTGCAGCGTTTCCTTCGTGCCTG 3017 AD-1334144 GGGCACCTTCCTCAATGACGCGG 3018 AD-1334145 TCCTGGAGAGGTGGTGCACGACG 3019 AD-1334146 CGCCGTGTGTTCATGTACGGGTG 3020 AD-1334147 AGCCTCTCTGCAGAAAAGCACAG 3021 AD-1334148 CTGGACTGCAGCAACAGCTCGGC 3022 AD-1334149 GGCTGTTTCAGCACACACTGCGT 3023 AD-1334150 GGCTGCATTGCCGAGGAGGACTG 3024 AD-1334151 GCCACCTACAAGCCTGGAGAGAC 3025 AD-1334152 GTCGACTGCAACACCTGCACCTG 3026 AD-1334153 AGGAACCGGAGGTGGGAGTGCAG 3027 AD-1334154 GATGGCCACTTCATCACCTTTGA 3028 AD-1334155 GGCGATCGCTACAGCTTTGAAGG 3029 AD-1334156 CAGCTGCGAGTACATCTTGGCCC 3030 AD-1334157 ACCTTCCGCATCGTCACCGAGAA 3031 AD-1334158 AGGCCATCAAGCTCTTCGTGGAG 3032 AD-1334159 GCTACGAGCTGATCCTCCAAGAG 3033 AD-1334160 GGGACCTTTAAGGCGGTGGCGAG 3034 AD-1334161 CCACCCTACAAGATACGCTACAT 3035 AD-1334162 TCTTCCTGGTCATCGAGACCCAC 3036 AD-1334163 GACCAGCGTGTTCATCCGACTGC 3037 AD-1334164 ACCAGGACTACAAGGGCAGGGTC 3038 AD-1334165 GCGGGAACTTCGACGACAATGCC 3039 AD-1334166 ATCAATGACTTTGCCACGCGTAG 3040 AD-1334167 ACGCACTGGAGTTTGGGAACAGC 3041 AD-1334168 TGGGCCCAGAAGCAGTGCAGCAT 3042 AD-1334169 TCCCAGGTTGACTCCACCAAGTA 3043 AD-1334170 TACGAGGCCTGCGTGAACGACGC 3044 AD-1334171 CGACTGCGAGTGTTTCTGCACGG 3045 AD-1334172 CCTGTGTGTGTCCTGGCGGACTC 3046 AD-1334173 CTTGTTCTGTGACTTCTACAACC 3047 AD-1334174 GCTGTGAGTGGCACTACCAGCCC 3048 AD-1334175 AGGCTGCTACCCGAAGTGCCCAC 3049 AD-1334176 CCAGCCCTTCTTCAATGAGGACC 3050 AD-1334177 TGAAGTGCGTGGCCCAGTGTGGC 3051 AD-1334178 TGCTACGACAAGGACGGAAACTA 3052 AD-1334179 TATGACGTCGGTGCAAGGGTCCC 3053 AD-1334180 TGCCAGAGCTGTAACTGCACACC 3054 AD-1334181 TCCAGTGCGCTCACAGCCTTGAG 3055 AD-1334182 ACCTGCACCTATGAGGACAGGAC 3056 AD-1334183 ACCAGGACGTCATCTACAACACC 3057 AD-1334184 GGCGCCTGCTTGATCGCCATCTG 3058 AD-1334185 GCACCATCATCAGGAAGGCTGTG 3059 AD-1334186 GCCACAACGCCATTCACCTTCAC 3060 AD-1334187 TCTCCACCGTGTGTGTCCGCGAG 3061 AD-1334188 GGTCCAGCTGGTACAATGGGCAC 3062 AD-1334189 GGCGGAGACTTTGAGACGTTTGA 3063 AD-1334190 GAGAGGGTACCAGGTATGCCCTG 3064 AD-1334191 TGCTGGCTGACATCGAGTGCCGG 3065 AD-1334192 AGCTTCCCGACATGCCGCTGGAG 3066 AD-1334193 AGCAGGTGGACTGTGACCGCATG 3067 AD-1334194 TGCGCCAACAGCCAACAGAGTCC 3068 AD-1334195 GCTCTGTCACGACTACGAGCTGC 3069 AD-1334196 GTTCTCTGCTGCGAATACGTGCC 3070 AD-1334197 AGCACGGAGCCTGCTGTGCCTAC 3071 AD-1334198 CCAGACCACAGCAACCGAAAAGA 1445 AD-1334199 CTCACCTCGCAGACTGGGTCCAG 3072 AD-1334200 GGACAGAGTGGTTTGATGAGGAC 3073 AD-1334201 GGGACGTTGAGTCCTACGATAAG 3074 AD-1334202 AGGGCCGCTGGAGGGCACTTATG 3075 AD-1334203 AGCAGCCTAAGGACATAGAGTGC 3076 AD-1334204 CCAACTGGACCCTGGCACAGGTG 3077 AD-1334205 AGGTGCACTGTGACGTCCACTTC 3078 AD-1334206 GTGTGCAGGAACTGGGAGCAGGA 3079 AD-1334207 GGCGTCTTCAAGATGTGCTACAA 3080 AD-1334208 CTCTGCTGCAGTGACGACCACTG 3081 AD-1334209 ACCGACCACAGAGCTGGAGACGG 3082 AD-1334210 AGGCCCTGTTCTCAACGCCGCAG 3083 AD-1334211 ACCCTCTCAGAAGGACTGACATC 3084 AD-1334212 CCCAGATACACAAGCACCCTTGG 3085 AD-1334213 AGGCTCCACAGAACCCACTGTCC 3086 AD-1334214 TCCACCCTTCCAACACGCTCAGC 3087 AD-1334215 CCCAACAACAATGGCAACCTCCA 3088 AD-1334216 ACCGCTTCCAAAGAGCCGCTGAC 3089 AD-1334217 TGGCGCCAACACTCACGAGCGAG 3090 AD-1334218 CTGTCCACCTCTCAGGCCGAGAC 3091 AD-1334219 CCCAGGACAGAGACGACAATGAG 3092 AD-1334220 CCCTTGACTAACACCACCACCAG 3093 AD-1334221 CGCTGTCAACCGAAGTGTGAGTG 3094 AD-1334222 ACAGAGTGGTTTGACGTGGACTT 3095 AD-1334223 ATGGAAACTTTTGAAAACATCAG 3096 AD-1334224 GGGCACCAAAGAGCATAGAGTGC 3097 AD-1334225 CCCGAGGTAAGCATCGACCAGGT 3098 AD-1334226 TGCTGACCTGCAGCCTGGAGACG 3099 AD-1334227 ACCTGCAAGAACGAAGACCAGAC 3100 AD-1334228 TGTGCTTCAACTACAACGTGCGT 3101 AD-1334229 CTTTGCTGTGACGACTACAGCCA 3102 AD-1334230 GGGACGACCTGGATCCTCACAAA 3103 AD-1334231 GCCGACCACAACAGCCACTACGA 3104 AD-1334232 CCTCCACCCTGAGAACAGCTCCC 3105 AD-1334233 CCTCCCAAAGTGCTGACCACCAC 3106 AD-1334234 ACCAGCTCCAAAGCCACTCCCTC 3107 AD-1334235 CTCCAGTCCAGGGACTGCAACCG 3108 AD-1334236 CCAGCACTGAGAAGCACAGCCAC 3109 AD-1334237 AGCTACCAGCGTTACACCCATCC 3110 AD-1334238 TCTTCCTCCCTGGGCACCACCTG 3111 AD-1334239 CGCCTATCACAGACCACCACACC 3112 AD-1334240 GGCCACCATGTCCACAGCCACAC 3113 AD-1334241 CCTCCTCCACTCCAGAGACTGCC 3114 AD-1334242 ACCTCCACAGTGCTTACCGCCAC 3115 AD-1334243 CCCAGGAACAGCTCACACTACCA 3116 AD-1334244 AGTGCCAACTACCACAACCACGG 3117 AD-1334245 CCTCCAGTGTGGATCAGCACAAC 3118 AD-1334246 ACACCCACAACCAGAGGCTCCAC 3119 AD-1334247 ACCGCCACAGTGCTGACCACCAC 3120 AD-1334248 TGGCCACTGGTTCTATGGCAACA 3121 AD-1334249 CCCTCCTCTAGCACACAGACCAG 3122 AD-1334250 ACGGCCACTACGATCACGGCCAC 3123 AD-1334251 CCCTCCTCAACTCCTGGGACAAC 3124 AD-1334252 ACCAGCAACACAGTGACTCCCTC 3125 AD-1334253 TCTGCCCTAGGGACCACCCACAC 3126 AD-1334254 CCAGTGCCGAACACCATGGCCAC 3127 AD-1334255 ACAGCCTGGACTTCGGCCACCTC 3128 AD-1334256 CCACCCACATCACAGAGCCTTCC 3129 AD-1334257 ACGGTGACTTCCCACACCCTAGC 3130 AD-1334258 AGCAACCACCGGTACCACCCAGC 3131 AD-1334259 CTCGACTCCAGCCCTTTCCAGCC 3132 AD-1334260 CCTAGCAGCAGAACCACCGAGTC 3133 AD-1334261 ACACCCAGCAAGACCCGCACCTC 3134 AD-1334262 CACGGTGGTGACCATGGGCTGTG 3135 AD-1334263 GAGTGGCTGGACTACAGCTACCC 3136 AD-1334264 CTTTGACACCTACTCCAACATCC 3137 AD-1334265 AGTTGGGCCAGGTCGTGGAATGC 3138 AD-1334266 AGCCTGGACTTTGGCCTGGTCTG 3139 AD-1334267 AGATGTGCTTCAACTATGAAATC 3140 AD-1334268 CGTGTGTTCTGCTGCAACTACGG 3141 AD-1334269 ACCAGCTCTACGGCCATGCCCTC 3142 AD-1334270 GGATCCTCACAGAGCTGACCACA 3143 AD-1334271 AGCCACTACGACTGAGTCCACTG 3144 AD-1334272 GGGACCACCTGGATCCTCACAGA 3145 AD-1334273 CCGAGCACTACAGCCACCGTGAC 3146 AD-1334274 CTCCTCCACCCAGGCAACTGCTG 3147 AD-1334275 ACGGCCACGACACCCACAGTCAC 3148 AD-1334276 CAGCTCCAAAGCCACTCCCTTCT 3149 AD-1334236 CCAGCACTGAGAAGCACAGCCAC 3109 AD-1334277 CCACAGCTACCAGCTTTACAGCC 3150 AD-1334239 CGCCTATCACAGACCACCACACC 3112 AD-1334240 GGCCACCATGTCCACAGCCACAC 3113 AD-1334278 CCTCCTCCACTCCAGAGACTGTC 3151 AD-1334279 CTCCACAGTGCTTACCACCACGG 3152 AD-1334280 CAGCTCACACTACCAAAGTGCTG 3153 AD-1334281 ACTACCACAACCACGGGCTTCAC 3154 AD-1334282 CGCACGCTTCCAGTGTGGATCAG 3155 AD-1334283 ACACCCACAACCAGAGGTTCCAC 3156 AD-1334284 GCTGACCACCACCACCACAACTG 3157 AD-1334248 TGGCCACTGGTTCTATGGCAACA 3121 AD-1334249 CCCTCCTCTAGCACACAGACCAG 3122 AD-1334250 ACGGCCACTACGATCACGGCCAC 3123 AD-1334285 CCCTCCTCAACTCCAGGGACAAC 3158 AD-1334286 ACCAGCAGCACAGTGACTCCCTC 3159 AD-1334253 TCTGCCCTAGGGACCACCCACAC 3126 AD-1334287 ACCACACACGGGCGATCCCTGTC 3160 AD-1334255 ACAGCCTGGACTTCGGCCACCTC 3128 AD-1334256 CCACCCACATCACAGAGCCTTCC 3129 AD-1334288 TACCACCCAGCACTCGACTCCAG 3161 AD-1334289 TCCAGCCCTCACCCTAGCAGCAG 3162 AD-1334261 ACACCCAGCAAGACCCGCACCTC 3134 AD-1334262 CACGGTGGTGACCATGGGCTGTG 3135 AD-1334263 GAGTGGCTGGACTACAGCTACCC 3136 AD-1334264 CTTTGACACCTACTCCAACATCC 3137 AD-1334265 AGTTGGGCCAGGTCGTGGAATGC 3138 AD-1334266 AGCCTGGACTTTGGCCTGGTCTG 3139 AD-1334267 AGATGTGCTTCAACTATGAAATC 3140 AD-1334268 CGTGTGTTCTGCTGCAACTACGG 3141 AD-1334290 ACCTGGATCCTCACAGAGCAGAC 3163 AD-1334291 AGCAGCCACTACGACCGCAACCA 3164 AD-1334292 CCTCCCAAAGTGCTGACCAGCAC 3165 AD-1334293 ACCAGTTCCAAAGCCACTTCCTC 3166 AD-1334294 TCCAAGGACTGCAACCACCCTTC 3167 AD-1334295 CAGTGCTGACAAGCACAGCCACC 3168 AD-1334296 TCCACAGCTACCAGCTTTACACC 3169 AD-1334297 CCCTCCTTCACCCTTGGGACCAC 3170 AD-1334298 CTCCCAGAACAGACCACCACACC 3171 AD-1334299 GCCACCATGTCCACAATCCACCC 3172 AD-1334300 ACCTCCACAGTGCTGACCACGAA 3173 AD-1334301 AGGGCCACCAGTTCCATGTCCAC 3174 AD-1334270 GGATCCTCACAGAGCTGACCACA 3143 AD-1334302 AGCCACTACAACTGCAGCCACTG 3175 AD-1334272 GGGACCACCTGGATCCTCACAGA 3145 AD-1334303 CCCAGCACTACAGCCACCGTGAC 3176 AD-1334304 GCACCCTCAAAGTGCTGACCAGC 3177 AD-1334305 ACACCCACAGTCATCAGCTCCAG 3178 AD-1334306 AGCCACTCCCTCCTCCAGTCCAG 3179 AD-1334236 CCAGCACTGAGAAGCACAGCCAC 3109 AD-1334307 AGCTACCAGCGTTACAGCCATCC 3180 AD-1334239 CGCCTATCACAGACCACCACACC 3112 AD-1334240 GGCCACCATGTCCACAGCCACAC 3113 AD-1334308 CTCCTCTACTCCAGAGACTGTCC 3181 AD-1334279 CTCCACAGTGCTTACCACCACGA 3182 AD-1334309 ACAGCTCACACTACCAAAGTGCC 3183 AD-1334310 GACTACCACAACCACGGGCTTCA 3184 AD-1334245 CCTCCAGTGTGGATCAGCACAAC 3118 AD-1334246 ACACCCACAACCAGAGGCTCCAC 3119 AD-1334247 ACCGCCACAGTGCTGACCACCAC 3120 AD-1334248 TGGCCACTGGTTCTATGGCAACA 3121 AD-1334249 CCCTCCTCTAGCACACAGACCAG 3122 AD-1334311 ACGGCCACTACGATCACAGCCAC 3185 AD-1334285 CCCTCCTCAACTCCAGGGACAAC 3158 AD-1334286 ACCAGCAGCACAGTGACTCCCTC 3159 AD-1334253 TCTGCCCTAGGGACCACCCACAC 3126 AD-1334312 AACACCACGGCCACCACACACGG 1551 AD-1334255 ACAGCCTGGACTTCGGCCACCTC 3128 AD-1334256 CCACCCACATCACAGAGCCTTCC 3129 AD-1334313 CCCAGCAGCAACCACCAGTACCA 3186 AD-1334289 TCCAGCCCTCACCCTAGCAGCAG 3162 AD-1334314 CACCTCCAGGACCACAGCCACAG 3187 AD-1334261 ACACCCAGCAAGACCCGCACCTC 3134 AD-1334315 TAACCACGGTGGTGACCACGGGC 3188 AD-1334263 GAGTGGCTGGACTACAGCTACCC 3136 AD-1334264 CTTTGACACCTACTCCAACATCC 3137 AD-1334265 AGTTGGGCCAGGTCGTGGAATGC 3138 AD-1334266 AGCCTGGACTTTGGCCTGGTCTG 3139 AD-1334267 AGATGTGCTTCAACTATGAAATC 3140 AD-1334268 CGTGTGTTCTGCTGCAACTACGG 3141 AD-1334316 TACGGCCACGCCCTCCTCAACTC 3189 AD-1334317 ACCTGGATCCTCACAAAGCTGAC 3190 AD-1334271 AGCCACTACGACTGAGTCCACTG 3144 AD-1334272 GGGACCACCTGGATCCTCACAGA 3145 AD-1334273 CCGAGCACTACAGCCACCGTGAC 3146 AD-1334274 CTCCTCCACCCAGGCAACTGCTG 3147 AD-1334275 ACGGCCACGACACCCACAGTCAC 3148 AD-1334276 CAGCTCCAAAGCCACTCCCTTCT 3149 AD-1334236 CCAGCACTGAGAAGCACAGCCAC 3109 AD-1334277 CCACAGCTACCAGCTTTACAGCC 3150 AD-1334239 CGCCTATCACAGACCACCACACC 3112 AD-1334240 GGCCACCATGTCCACAGCCACAC 3113 AD-1334241 CCTCCTCCACTCCAGAGACTGCC 3114 AD-1334279 CTCCACAGTGCTTACCACCACGG 3152 AD-1334309 ACAGCTCACACTACCAAAGTGCC 3183 AD-1334310 GACTACCACAACCACGGGCTTCA 3184 AD-1334245 CCTCCAGTGTGGATCAGCACAAC 3118 AD-1334318 ACACCCACAACCAGTGGCTCCAC 3191 AD-1334284 GCTGACCACCACCACCACAACTG 3157 AD-1334248 TGGCCACTGGTTCTATGGCAACA 3121 AD-1334249 CCCTCCTCTAGCACACAGACCAG 3122 AD-1334250 ACGGCCACTACGATCACGGCCAC 3123 AD-1334285 CCCTCCTCAACTCCAGGGACAAC 3158 AD-1334286 ACCAGCAGCACAGTGACTCCCTC 3159 AD-1334253 TCTGCCCTAGGGACCACCCACAC 3126 AD-1334287 ACCACACACGGGCGATCCCTGTC 3160 AD-1334255 ACAGCCTGGACTTCGGCCACCTC 3128 AD-1334256 CCACCCACATCACAGAGCCTTCC 3129 AD-1334288 TACCACCCAGCACTCGACTCCAG 3161 AD-1334289 TCCAGCCCTCACCCTAGCAGCAG 3162 AD-1334261 ACACCCAGCAAGACCCGCACCTC 3134 AD-1334315 TAACCACGGTGGTGACCACGGGC 3188 AD-1334263 GAGTGGCTGGACTACAGCTACCC 3136 AD-1334264 CTTTGACACCTACTCCAACATCC 3137 AD-1334265 AGTTGGGCCAGGTCGTGGAATGC 3138 AD-1334266 AGCCTGGACTTTGGCCTGGTCTG 3139 AD-1334267 AGATGTGCTTCAACTATGAAATC 3140 AD-1334268 CGTGTGTTCTGCTGCAACTACGG 3141 AD-1334269 ACCAGCTCTACGGCCATGCCCTC 3142 AD-1334319 GGGACGACCTGGATCCTCACAGA 3192 AD-1334320 GCTGACCACAACAGCCACTACGA 3193 AD-1334321 GCATCCACTGGATCCACGGCCAC 3194 AD-1334322 CCCTCCCAAAGTGCTGACCAGCC 3195 AD-1334293 ACCAGTTCCAAAGCCACTTCCTC 3166 AD-1334294 TCCAAGGACTGCAACCACCCTTC 3167 AD-1334323 AGCCACCAAATCCACAGCTACCA 3196 AD-1334298 CTCCCAGAACAGACCACCACACC 3171 AD-1334299 GCCACCATGTCCACAATCCACCC 3172 AD-1334300 ACCTCCACAGTGCTGACCACGAA 3173 AD-1334324 AGGGCCACCAGTTCCACGTCCAC 3197 AD-1334270 GGATCCTCACAGAGCTGACCACA 3143 AD-1334302 AGCCACTACAACTGCAGCCACTG 3175 AD-1334272 GGGACCACCTGGATCCTCACAGA 3145 AD-1334320 GCTGACCACAACAGCCACTACGA 3193 AD-1334272 GGGACCACCTGGATCCTCACAGA 3145 AD-1334273 CCGAGCACTACAGCCACCGTGAC 3146 AD-1334274 CTCCTCCACCCAGGCAACTGCTG 3147 AD-1334325 TGTGAGCACCACGGCCACGACAC 3198 AD-1334234 ACCAGCTCCAAAGCCACTCCCTC 3107 AD-1334326 GTCCAGGGACTGCAACTGCCCTT 3199 AD-1334236 CCAGCACTGAGAAGCACAGCCAC 3109 AD-1334277 CCACAGCTACCAGCTTTACAGCC 3150 AD-1334239 CGCCTATCACAGACCACCACACC 3112 AD-1334240 GGCCACCATGTCCACAGCCACAC 3113 AD-1334278 CCTCCTCCACTCCAGAGACTGTC 3151 AD-1334242 ACCTCCACAGTGCTTACCGCCAC 3115 AD-1334309 ACAGCTCACACTACCAAAGTGCC 3183 AD-1334310 GACTACCACAACCACGGGCTTCA 3184 AD-1334245 CCTCCAGTGTGGATCAGCACAAC 3118 AD-1334318 ACACCCACAACCAGTGGCTCCAC 3191 AD-1334327 ACCGCCAGAGTGCTGACCACCAC 3200 AD-1334248 TGGCCACTGGTTCTATGGCAACA 3121 AD-1334249 CCCTCCTCTAGCACACAGACCAG 3122 AD-1334250 ACGGCCACTACGATCACGGCCAC 3123 AD-1334328 TCCAGGGACAACACCCATCACCC 3201 AD-1334329 AGCTCCAAAGCCACTTCCTCCTC 3202 AD-1334294 TCCAAGGACTGCAACCACCCTTC 3167 AD-1334330 AGTGCTGACAAGCACAGCCACAA 3203 AD-1334296 TCCACAGCTACCAGCTTTACACC 3169 AD-1334331 TCCTCCACCCTGTGGACCACGTG 3204 AD-1334332 GTCCCAGCACAGACCACCACACC 3205 AD-1334333 ATGTCCACCATGTCCACAATCCA 3206 AD-1334334 ACCTCCTCTACTCCAGAGACCAC 3207 AD-1334335 ACCTCCACAGTGCTGACCACCAC 3208 AD-1334336 AGGGCCACCAATTCCACGGCCAC 3209 AD-1334337 GCTGACCACAACAGCCACTACAA 3210 AD-1334338 ACTGGATCCACGGCCACCCTGTC 3211 AD-1334272 GGGACCACCTGGATCCTCACAGA 3145 AD-1334339 CCGAGCACTATAGCCACCGTGAT 3212 AD-1334340 CTCCACTCTGGGAACAGCTCACA 3213 AD-1334341 ACCATGGCCACTATGCCCACAGC 3214 AD-1334342 ACTGCCTCCACGGTTCCCAGCTC 3215 AD-1334343 CTGCCAACCTTCAGCGTGTCCAC 3216 AD-1334344 TGTCCTCCTCAGTCCTCACCACC 3217 AD-1334345 GCTCCCACTTCTCTACTCCCTGC 3218 AD-1334346 GGGCATTTGGACAGTTTTTCTCG 3219 AD-1334347 GGGAAGTCATCTACAATAAGACC 3220 AD-1334348 GGCTGCCATTTCTACGCAGTGTG 3221 AD-1334349 AGCACTGTGACATTGACCGCTTC 3222 AD-1334350 GCTGTGACAATGCCATCCCTCTC 3223 AD-1334351 GGACCCTGGAGAACTGCACGGTG 3224 AD-1334352 GCGTGGGTGACAACCGTGTCGTC 3225 AD-1334353 TGGACCCAAAGCCTGTGGCCAAC 3226 AD-1334354 ACCTGCGTGAACAAGCACCTGCC 3227 AD-1334355 CATCAAAGTGTCGGACCCGAGCC 3228 AD-1334356 GCCCTGTGACTTCCACTATGAGT 3229 AD-1334357 GCGAGTGCATCTGCAGCATGTGG 3230 AD-1334358 CTCCCACTATTCCACCTTTGACG 3231 AD-1334359 GCACCTATGTCCTCATGAGAGAG 3232 AD-1334360 TGCACGCTTTGGGAATCTCAGCC 3233 AD-1334361 ACCTGGACAACCACTACTGCACG 3234 AD-1334362 CCCTCAGCATCCACTACAAGTCC 3235 AD-1334363 TCGTCCTCACTGTCACCATGGTG 3236 AD-1334364 GGCCTGATCCTGTTTGACCAAAT 3237 AD-1334365 GCAGCGGTTTCAGCAAGAACGGC 3238 AD-1334366 TGCGTGTGGACATTCCTGCCCTG 3239 AD-1334367 GCGTGAGCGTCACCTTCAATGGC 3240 AD-1334368 ACAGCCTCTTCCACAACAACACC 3241 AD-1334369 CCTGCACCAACAACCAGAGGGAC 3242 AD-1334370 ACTGTCTCCAGCGGGACGGAACC 3243 AD-1334371 GCCGCCAGTTGCAAGGACATGGC 3244 AD-1334372 CCCGACAGCAGAAAGGATGGCTG 3245 AD-1334373 AGCCGCTCTGTGATCTGATGCTG 3246 AD-1334374 GCCAGGTCTTTGCTGAGTGCCAC 3247 AD-1334375 CCGGGCCCATTCTTCAACGCCTG 3248 AD-1334376 TGGAGGCTTACGCAGAGCTCTGC 3249 AD-1334377 GGAGTGTGCAGTGACTGGCGAGG 3250 AD-1334378 ACCCACCAAAGTGTACAAGCCAT 3251 AD-1334379 ACCTGCAACTCTAGGAACCAGAG 3252 AD-1334380 ACCAGATCCTCTTCAACGCACAC 3253 AD-1334381 ATGGGCATCTGCGTGCAGGCCTG 3254 AD-1334382 CCCGATGGGTTTCCTAAATTTCC 3255 AD-1334383 TGGGTCAGCAACTGCCAGTCCTG 3256 AD-1334384 ACGAGGGTTCAGTGTCGGTGCAG 3257 AD-1334385 CCCGGCTTCGTAACCGTGACCAG 3258 AD-1334386 TGCGTGTGCAACACAACCACCTG 3259 AD-1334387 GGCAGGAGTCCATCTGCACCCAG 3260 AD-1334388 TGCTGTCCCACCTTCCGCTGCAG 3261 AD-1334389 CCTCAGCTGTGTTCGTACAATGG 3262 AD-1334390 GGTTGGTGCAACCTTCCCAGGCG 3263 AD-1334391 CTTCCCTGCCACATGTGTACCTG 3264 AD-1334392 CAACGGTGCAATGTCAGGAGGAT 3265 AD-1334393 GCCTGCAACAATACTACCTGTCC 3266 AD-1334394 CAGGGCTTTGAGTACAAGAGAGT 3267 AD-1334395 CAGTCCAGCTGAATGAAACCTGG 3268 AD-1334396 GTCAACAGCCATGTGGACAACTG 3269 AD-1334397 CACCGTGTACCTCTGTGAGGCTG 3270 AD-1334398 AGGGTGGAGTCCATTTGCTGACC 3271 AD-1334399 TCCTGCCCAGATGTGTCCAGCTG 3272 AD-1334400 CTGCTGCTACTCCTGTGAGGAGG 3273 AD-1334401 ACTCCTGTCAAGTCCGCATCAAC 3274 AD-1334402 ACGACCATCCTGTGGCACCAGGG 3275 AD-1334403 GAGGTCAACATCACCTTCTGCGA 3276 AD-1334404 AGCGTCCAAGTACTCAGCAGAGG 3277 AD-1334405 CCATGCAGCACCAGTGCACCTGC 3278 AD-1334406 GTGCCCTTGCACTGTCCTAACGG 3279 AD-1334407 TCCTGCACACCTACACCCACGTG 3280 AD-1334408 CTGCACGCCCTTCTGTGTCCCTG 3281 AD-1334409 CCACTGCTGTCTGAGAACGTTCT 3282 AD-1334410 CCCATGCTCTGTCCACCTGGAGC 3283 AD-1334411 GTGCATTGTCTGATCATGAAAAC 3284 AD-1334412 AGGGCGCCACTCAGGAGTCCTAC 3285 AD-1334413 CCCTCCCTGATGTCACTGGGACG 3286 AD-1334414 CCCTGGAACAAACTAAGCATGTG 3287 AD-1334415 CAGCACGGATTCCAGCTGGCCAC 3288 AD-1334416 CAGACAGGCTGGTCCAGGCAAGG 3289 AD-1334417 CTGCTGCCAGGAAGCTGCGACAG 3290 AD-1334418 CTGCAGGGTAACTCAGGGCTGAG 3291 AD-1334419 TCGCAACGGCCAGGTCAGAGAGG 3292 AD-1334420 CCAGCCCAGTTTTGCAAATAAAC 3293

TABLE 6 Unmodified Sense and Antisense Strand MUC5B dsRNA Sequences SEQ Duplex SEQ ID ID Start Site in End Site in Name Sense Sequence 5′ to 3′ NO: Antisense Sequence 5′ to 3′ NO: NM_002458.3 NM_002458.3 AD-1334421 UUGGCUCUGGCGGCCAUGCUC 3294 GAGCAUGGCCGCCAGAGCCAACA 3581 90 112 AD-1334422 CUGGGAGAAUGCAGGGCACAC 3295 GUGUGCCCUGCAUUCUCCCAGCU 3582 152 174 AD-1334423 GCGCGUGAGCUUUGUUCCACC 3296 GGUGGAACAAAGCUCACGCGCCG 3583 209 231 AD-1334424 UGGGCGGGUGUGCAGCACCUG 3297 CAGGUGCUGCACACCCGCCCAUU 3584 275 297 AD-1334425 CCACUACAAGACCUUCGACGG 3298 CCGUCGAAGGUCUUGUAGUGGAA 3585 305 327 AD-1334426 CCUUUGCAACUACGUGUUCUC 3299 GAGAACACGUAGUUGCAAAGGCC 3586 347 369 AD-1334427 GAGGACUUCAACGUCCAGCUA 1681 UAGCUGGACGUUGAAGUCCUCGU 2005 390 412 AD-1334428 ACCCGUGUUGUCAUCAAGGCC 3300 GGCCUUGAUGACAACACGGGUGA 3587 444 466 AD-1334429 GGCUCCGUCCUCAUCAAUGGG 3301 CCCAUUGAUGAGGACGGAGCCGU 3588 492 514 AD-1334430 GCUGCCUUACAGCCGCACUGG 3302 CCAGUGCGGCUGUAAGGCAGCUC 3589 524 546 AD-1334431 GACUACAUCAAGGUCAGCAUC 3303 GAUGCUGACCUUGAUGUAGUCCC 3590 567 589 AD-1334432 GCUGACAUUCCUGUGGAACGG 3304 CCGUUCCACAGGAAUGUCAGCAC 3591 596 618 AD-1334433 CUGGAUCCCAAAUACGCCAAC 3305 GUUGGCGUAUUUGGGAUCCAGCU 3592 639 661 AD-1334434 GCCUUCAACGAGUUCUAUGCC 3306 GGCAUAGAACUCGUUGAAGGCCG 3593 699 721 AD-1334435 AACCUGCAGAAGUUGGAUGGG 3307 CCCAUCCAACUUCUGCAGGUUCC 3594 753 775 AD-1334436 UGCACGGACGAGGAGGGCAUC 3308 GAUGCCCUCCUCGUCCGUGCAGU 3595 819 841 AD-1334437 UUUGCGGAGUGCCACGCACUG 3309 CAGUGCGUGGCACUCCGCAAAGG 3596 867 889 AD-1334438 GACAGCACUGCGUACCUGGCC 3310 GGCCAGGUACGCAGUGCUGUCCA 3597 891 913 AD-1334439 GCCACCUUUGUGGAAUACUCA 1693 UGAGUAUUCCACAAAGGUGGCAC 2017 954 976 AD-1334440 CUGGAGGUGCCCUGAGCUCUG 3311 CAGAGCUCAGGGCACCUCCAGUU 3598 1010 1032 AD-1334441 CUCAACAUGCAGCACCAGGAG 3312 CUCCUGGUGCUGCAUGUUGAGGG 3599 1047 1069 AD-1334442 ACCCUGCACGGACACCUGCUC 3313 GAGCAGGUGUCCGUGCAGGGUGA 3600 1076 1098 AD-1334443 GGACCACUGUGUGGACGGCUG 3314 CAGCCGUCCACACAGUGGUCCUC 3601 1124 1146 AD-1334444 GCUGGAUGACAUCACGCACUC 3315 GAGUGCGUGAUGUCAUCCAGCAC 3602 1166 1188 AD-1334445 CUCCUUCAACACCACCUGCAG 3316 CUGCAGGUGGUGUUGAAGGAGGU 3603 1250 1272 AD-1334446 CUAUGGCAGUGCCAGGACCUG 3317 CAGGUCCUGGCACUGCCAUAGCC 3604 1293 1315 AD-1334447 ACCUAUGAUGAGAAACUCUAC 3318 GUAGAGUUUCUCAUCAUAGGUGG 3605 1359 1381 AD-1334448 AGCUACGUUCUGUCCAAGAAA 1702 UUUCUUGGACAGAACGUAGCUGC 2026 1398 1420 AD-1334449 GACAGCAGCUUCACCGUGCUG 3319 CAGCACGGUGAAGCUGCUGUCGG 3606 1425 1447 AD-1334450 GGACAACGAGAACUGCCUGAA 1704 UUCAGGCAGUUCUCGUUGUCCGU 2028 1472 1494 AD-1334451 UCCUCAACUCCAUCUACACGC 3320 GCGUGUAGAUGGAGUUGAGGAAC 3607 1558 1580 AD-1334452 GCCAACAUCACCCUGUUCACA 1706 UGUGAACAGGGUGAUGUUGGCUG 2030 1596 1618 AD-1334453 UCGAGCUUCUUCAUCGUGGUG 3321 CACCACGAUGAAGAAGCUCGAGG 3608 1620 1642 AD-1334454 GCCACUCAUGCAGGUGUUUGU 3322 ACAAACACCUGCAUGAGUGGCAC 3609 1679 1701 AD-1334455 GGGAACUUCAACCAGAACCAG 3323 CUGGUUCUGGUUGAAGUUCCCAC 3610 1743 1765 AD-1334456 UGACGACUUCACGGCCCUCAG 3324 CUGAGGGCCGUGAAGUCGUCAGC 3611 1766 1788 AD-1334457 AGCCUUCGCCAACACCUGGAA 1711 UUCCAGGUGUUGGCGAAGGCUGC 2035 1811 1833 AD-1334458 CCAGGAACAGCUUUGAGGACC 3325 GGUCCUCAAAGCUGUUCCUGGCA 3612 1855 1877 AD-1334459 GUGGAGAAUGAGAACUACGCC 3326 GGCGUAGUUCUCAUUCUCCACAC 3613 1890 1912 AD-1334460 CAACAGUGCCUUCUCGCGCUG 3327 CAGCGCGAGAAGGCACUGUUGGG 3614 1940 1962 AD-1334461 CUUCCACUCGAACUGCAUGUU 58 AACAUGCAGUUCGAGUGGAAGGG 389 1985 2007 AD-1334462 CACCUGCAACUGUGAGCGGAG 3328 CUCCGCUCACAGUUGCAGGUGUC 3615 2009 2031 AD-1334463 CCUCCUAUGUGCACGCCUGUG 3329 CACAGGCGUGCACAUAGGAGGAC 3616 2056 2078 AD-1334464 GGCGUACAGCUCAGCGACUGG 3330 CCAGUCGCUGAGCUGUACGCCCU 3617 2085 2107 AD-1334465 ACCAAGUACAUGCAGAACUGC 3331 GCAGUUCUGCAUGUACUUGGUGC 3618 2121 2143 AD-1334466 UACGCCUACGUGGUGGAUGCC 3332 GGCAUCCACCACGUAGGCGUAGC 3619 2157 2179 AD-1334467 GCAGCGUUUCCUUCGUGCCUG 3333 CAGGCACGAAGGAAACGCUGCAG 3620 2221 2243 AD-1334468 GCACCUUCCUCAAUGACGCGG 3334 CCGCGUCAUUGAGGAAGGUGCCC 3621 2266 2288 AD-1334469 CUGGAGAGGUGGUGCACGACG 3335 CGUCGUGCACCACCUCUCCAGGA 3622 2344 2366 AD-1334470 CCGUGUGUUCAUGUACGGGUG 3336 CACCCGUACAUGAACACACGGCG 3623 2371 2393 AD-1334471 CCUCUCUGCAGAAAAGCACAG 3337 CUGUGCUUUUCUGCAGAGAGGCU 3624 2413 2435 AD-1334472 GGACUGCAGCAACAGCUCGGC 3338 GCCGAGCUGUUGCUGCAGUCCAG 3625 2459 2481 AD-1334473 CUGUUUCAGCACACACUGCGU 3339 ACGCAGUGUGUGCUGAAACAGCC 3626 2531 2553 AD-1334474 CUGCAUUGCCGAGGAGGACUG 3340 CAGUCCUCCUCGGCAAUGCAGCC 3627 2600 2622 AD-1334475 CACCUACAAGCCUGGAGAGAC 334 GUCUCUCCAGGCUUGUAGGUGGC 3628 2642 2664 AD-1334476 CGACUGCAACACCUGCACCUG 3342 CAGGUGCAGGUGUUGCAGUCGAC 3629 2672 2694 AD-1334477 GAACCGGAGGUGGGAGUGCAG 3343 CUGCACUCCCACCUCCGGUUCCU 3630 2696 2718 AD-1334478 UGGCCACUUCAUCACCUUUGA 1732 UCAAAGGUGAUGAAGUGGCCAUC 2056 2756 2778 AD-1334479 CGAUCGCUACAGCUUUGAAGG 3344 CCUUCAAAGCUGUAGCGAUCGCC 3631 2780 2802 AD-1334480 GCUGCGAGUACAUCUUGGCCC 3345 GGGCCAAGAUGUACUCGCAGCUG 3632 2803 2825 AD-1334481 CUUCCGCAUCGUCACCGAGAA 1735 UUCUCGGUGACGAUGCGGAAGGU 2059 2858 2880 AD-1334482 GCCAUCAAGCUCUUCGUGGAG 3346 CUCCACGAAGAGCUUGAUGGCCU 3633 2916 2938 AD-1334483 UACGAGCUGAUCCUCCAAGAG 3347 CUCUUGGAGGAUCAGCUCGUAGC 3634 2940 2962 AD-1334484 GACCUUUAAGGCGGUGGCGAG 3348 CUCGCCACCGCCUUAAAGGUCCC 3635 2963 2985 AD-1334485 ACCCUACAAGAUACGCUACAU 3349 AUGUAGCGUAUCUUGUAGGGUGG 3636 3002 3024 AD-1334486 UUCCUGGUCAUCGAGACCCAC 3350 GUGGGUCUCGAUGACCAGGAAGA 3637 3030 3052 AD-1334487 CCAGCGUGUUCAUCCGACUGC 3351 GCAGUCGGAUGAACACGCUGGUC 3638 3079 3101 AD-1334488 CAGGACUACAAGGGCAGGGUC 3352 GACCCUGCCCUUGUAGUCCUGGU 3639 3102 3124 AD-1334489 GGGAACUUCGACGACAAUGCC 3353 GGCAUUGUCGUCGAAGUUCCCGC 3640 3135 3157 AD-1334490 CAAUGACUUUGCCACGCGUAG 3354 CUACGCGUGGCAAAGUCAUUGAU 3641 3158 3180 AD-1334491 GCACUGGAGUUUGGGAACAGC 3355 GCUGUUCCCAAACUCCAGUGCGU 3642 3198 3220 AD-1334492 GGCCCAGAAGCAGUGCAGCAU 3356 AUGCUGCACUGCUUCUGGGCCCA 3643 3296 3318 AD-1334493 CCAGGUUGACUCCACCAAGUA 174 UACUUGGUGGAGUCAACCUGGGA 2071 3350 3372 AD-1334494 CGAGGCCUGCGUGAACGACGC 3357 GCGUCGUUCACGCAGGCCUCGUA 3644 3374 3396 AD-1334495 ACUGCGAGUGUUUCUGCACGG 3358 CCGUGCAGAAACACUCGCAGUCG 3645 3418 3440 AD-1334496 UGUGUGUGUCCUGGCGGACUC 3359 GAGUCCGCCAGGACACACACAGG 3646 3478 3500 AD-1334497 UGUUCUGUGACUUCUACAACC 3360 GGUUGUAGAAGUCACAGAACAAG 3647 3514 3536 AD-1334498 UGUGAGUGGCACUACCAGCCC 3361 GGGCUGGUAGUGCCACUCACAGC 3648 3546 3568 AD-1334499 GCUGCUACCCGAAGUGCCCAC 3362 GUGGGCACUUCGGGUAGCAGCCU 3649 3640 3662 AD-1314302 AGCCCUUCUUCAAUGAGGACC 3363 GGUCCUCAUUGAAGAAGGGCUGG 3650 3667 3689 AD-1334500 AAGUGCGUGGCCCAGUGUGGC 3364 GCCACACUGGGCCACGCACUUCA 3651 3693 3715 AD-1334501 CUACGACAAGGACGGAAACUA 1756 UAGUUUCCGUCCUUGUCGUAGCA 2080 3716 3738 AD-1334502 UGACGUCGGUGCAAGGGUCCC 3365 GGGACCCUUGCACCGACGUCAUA 3652 3740 3762 AD-1334503 CCAGAGCUGUAACUGCACACC 3366 GGUGUGCAGUUACAGCUCUGGCA 3653 3776 3798 AD-1334504 CAGUGCGCUCACAGCCUUGAG 3367 CUCAAGGCUGUGAGCGCACUGGA 3654 3807 3829 AD-1334505 CUGCACCUAUGAGGACAGGAC 3368 GUCCUGUCCUCAUAGGUGCAGGU 3655 3836 3858 AD-1334506 CAGGACGUCAUCUACAACACC 3369 GGUGUUGUAGAUGACGUCCUGGU 3656 3867 3889 AD-1334507 CGCCUGCUUGAUCGCCAUCUG 3370 CAGAUGGCGAUCAAGCAGGCGCC 3657 3902 3924 AD-1334508 ACCAUCAUCAGGAAGGCUGUG 3371 CACAGCCUUCCUGAUGAUGGUGC 3658 3936 3958 AD-1334509 CACAACGCCAUUCACCUUCAC 3372 GUGAAGGUGAAUGGCGUUGUGGC 3659 3977 3999 AD-1334510 UCCACCGUGUGUGUCCGCGAG 3373 CUCGCGGACACACACGGUGGAGA 3660 4044 4066 AD-1334511 UCCAGCUGGUACAAUGGGCAC 3374 GUGCCCAUUGUACCAGCUGGACC 3661 4077 4099 AD-1334512 CGGAGACUUUGAGACGUUUGA 1767 UCAAACGUCUCAAAGUCUCCGCC 2091 4121 4143 AD-1334513 GAGGGUACCAGGUAUGCCCUG 3375 CAGGGCAUACCUGGUACCCUCUC 3662 4156 4178 AD-1334514 CUGGCUGACAUCGAGUGCCGG 3376 CCGGCACUCGAUGUCAGCCAGCA 3663 4179 4201 AD-1334515 CUUCCCGACAUGCCGCUGGAG 3377 CUCCAGCGGCAUGUCGGGAAGCU 3664 4209 4231 AD-1334516 CAGGUGGACUGUGACCGCAUG 3378 CAUGCGGUCACAGUCCACCUGCU 3665 4242 4264 AD-1334517 CGCCAACAGCCAACAGAGUCC 3379 GGACUCUGUUGGCUGUUGGCGCA 3666 4277 4299 AD-1334518 UCUGUCACGACUACGAGCUGC 3380 GCAGCUCGUAGUCGUGACAGAGC 3667 4303 4325 AD-1334519 UCUCUGCUGCGAAUACGUGCC 3381 GGCACGUAUUCGCAGCAGAGAAC 3668 4328 4350 AD-1334520 CACGGAGCCUGCUGUGCCUAC 3382 GUAGGCACAGCAGGCUCCGUGCU 3669 4403 4425 AD-1334521 AGACCACAGCAACCGAAAAGA 1776 UCUUUUCGGUUGCUGUGGUCUGG 2100 4432 4454 AD-1334522 CACCUCGCAGACUGGGUCCAG 3383 CUGGACCCAGUCUGCGAGGUGAG 3670 4496 4518 AD-1334523 ACAGAGUGGUUUGAUGAGGAC 3384 GUCCUCAUCAAACCACUCUGUCC 3671 4584 4606 AD-1334524 GACGUUGAGUCCUACGAUAAG 3385 CUUAUCGUAGGACUCAACGUCCC 3672 4632 4654 AD-1334525 GGCCGCUGGAGGGCACUUAUG 3386 CAUAAGUGCCCUCCAGCGGCCCU 3673 4658 4680 AD-1334526 CAGCCUAAGGACAUAGAGUGC 3387 GCACUCUAUGUCCUUAGGCUGCU 3674 4683 4705 AD-1334527 AACUGGACCCUGGCACAGGUG 3388 CACCUGUGCCAGGGUCCAGUUGG 3675 4722 4744 AD-1334528 GUGCACUGUGACGUCCACUUC 3389 GAAGUGGACGUCACAGUGCACCU 3676 4752 4774 AD-1334529 GUGCAGGAACUGGGAGCAGGA 1784 UCCUGCUCCCAGUUCCUGCACAC 2108 4781 4803 AD-1334530 CGUCUUCAAGAUGUGCUACAA 1785 UUGUAGCACAUCUUGAAGACGCC 2109 4805 4827 AD-1334531 CUGCUGCAGUGACGACCACUG 3390 CAGUGGUCGUCACUGCAGCAGAG 3677 4844 4866 AD-1334532 CGACCACAGAGCUGGAGACGG 3391 CCGUCUCCAGCUCUGUGGUCGGU 3678 4891 4913 AD-1334533 GCCCUGUUCUCAACGCCGCAG 3392 CUGCGGCGUUGAGAACAGGGCCU 3679 4932 4954 AD-1334534 CCUCUCAGAAGGACUGACAUC 3393 GAUGUCAGUCCUUCUGAGAGGGU 3680 5012 5034 AD-1334535 CAGAUACACAAGCACCCUUGG 3394 CCAAGGGUGCUUGUGUAUCUGGG 3681 5036 5058 AD-1334536 GCUCCACAGAACCCACUGUCC 3395 GGACAGUGGGUUCUGUGGAGCCU 3682 5092 5114 AD-1334537 CACCCUUCCAACACGCUCAGC 3396 GCUGAGCGUGUUGGAAGGGUGGA 3683 5129 5151 AD-1334538 CAACAACAAUGGCAACCUCCA 1793 UGGAGGUUGCCAUUGUUGUUGGG 2117 5212 5234 AD-1334539 CGCUUCCAAAGAGCCGCUGAC 3397 GUCAGCGGCUCUUUGGAAGCGGU 3684 5261 5283 AD-1334540 GCGCCAACACUCACGAGCGAG 3398 CUCGCUCGUGAGUGUUGGCGCCA 3685 5292 5314 AD-1334541 GUCCACCUCUCAGGCCGAGAC 3399 GUCUCGGCCUGAGAGGUGGACAG 3686 5315 5337 AD-1334542 CAGGACAGAGACGACAAUGAG 3400 CUCAUUGUCGUCUCUGUCCUGGG 3687 5345 5367 AD-1334543 CUUGACUAACACCACCACCAG 3401 CUGGUGGUGGUGUUAGUCAAGGG 3688 5369 5391 AD-1334544 CUGUCAACCGAAGUGUGAGUG 3402 CACUCACACUUCGGUUGACAGCG 3689 5405 5427 AD-1334545 AGAGUGGUUUGACGUGGACUU 3403 AAGUCCACGUCAAACCACUCUGU 3690 5429 5451 AD-1334546 GGAAACUUUUGAAAACAUCAG 3404 CUGAUGUUUUCAAAAGUUUCCAU 3691 5480 5502 AD-1334547 GCACCAAAGAGCAUAGAGUGC 3405 GCACUCUAUGCUCUUUGGUGCCC 3692 5526 5548 AD-1334548 CGAGGUAAGCAUCGACCAGGU 3406 ACCUGGUCGAUGCUUACCUCGGG 3693 5564 5586 AD-1334549 CUGACCUGCAGCCUGGAGACG 3407 CGUCUCCAGGCUGCAGGUCAGCA 3694 5595 5617 AD-1334550 CUGCAAGAACGAAGACCAGAC 3408 GUCUGGUCUUCGUUCUUGCAGGU 3695 5624 5646 AD-1334551 UGCUUCAACUACAACGUGCGU 3409 ACGCACGUUGUAGUUGAAGCACA 3696 5661 5683 AD-1334552 UUGCUGUGACGACUACAGCCA 1807 UGGCUGUAGUCGUCACAGCAAAG 2131 5687 5709 AD-1334553 GACGACCUGGAUCCUCACAAA 1808 UUUGUGAGGAUCCAGGUCGUCCC 2132 5762 5784 AD-1334554 CGACCACAACAGCCACUACGA 1809 UCGUAGUGGCUGUUGUGGUCGGC 2133 5785 5807 AD-1334555 UCCACCCUGAGAACAGCUCCC 3410 GGGAGCUGUUCUCAGGGUGGAGG 3697 5838 5860 AD-1334556 UCCCAAAGUGCUGACCACCAC 3411 GUGGUGGUCAGCACUUUGGGAGG 3698 5861 5883 AD-1334557 CAGCUCCAAAGCCACUCCCUC 3412 GAGGGAGUGGCUUUGGAGCUGGU 3699 5903 5925 AD-1334558 CCAGUCCAGGGACUGCAACCG 3413 CGGUUGCAGUCCCUGGACUGGAG 3700 5926 5948 AD-1334559 AGCACUGAGAAGCACAGCCAC 3414 GUGGCUGUGCUUCUCAGUGCUGG 3701 5954 5976 AD-1334560 CUACCAGCGUUACACCCAUCC 3415 GGAUGGGUGUAACGCUGGUAGCU 3702 5986 6008 AD-1334561 UUCCUCCCUGGGCACCACCUG 3416 CAGGUGGUGCCCAGGGAGGAAGA 3703 6011 6033 AD-1334562 CCUAUCACAGACCACCACACC 3417 GGUGUGGUGGUCUGUGAUAGGCG 3704 6038 6060 AD-1334563 CCACCAUGUCCACAGCCACAC 3418 GUGUGGCUGUGGACAUGGUGGCC 3705 6064 6086 AD-1334564 UCCUCCACUCCAGAGACUGCC 3419 GGCAGUCUCUGGAGUGGAGGAGG 3706 6087 6109 AD-1334565 CUCCACAGUGCUUACCGCCAC 3420 GUGGCGGUAAGCACUGUGGAGGU 3707 6113 6135 AD-1334566 CAGGAACAGCUCACACUACCA 1821 UGGUAGUGUGAGCUGUUCCUGGG 2145 6184 6206 AD-1334567 UGCCAACUACCACAACCACGG 3421 CCGUGGUUGUGGUAGUUGGCACU 3708 6208 6230 AD-1334568 UCCAGUGUGGAUCAGCACAAC 3422 GUUGUGCUGAUCCACACUGGAGG 3709 6275 6297 AD-1334569 ACCCACAACCAGAGGCUCCAC 3423 GUGGAGCCUCUGGUUGUGGGUGU 3710 6302 6324 AD-1334570 CGCCACAGUGCUGACCACCAC 3424 GUGGUGGUCAGCACUGUGGCGGU 3711 6359 6381 AD-1334571 GCCACUGGUUCUAUGGCAACA 1826 UGUUGCCAUAGAACCAGUGGCCA 2150 6393 6415 AD-1334572 CUCCUCUAGCACACAGACCAG 3425 CUGGUCUGUGUGCUAGAGGAGGG 3712 6416 6438 AD-1334573 GGCCACUACGAUCACGGCCAC 3426 GUGGCCGUGAUCGUAGUGGCCGU 3713 6464 6486 AD-1334574 CUCCUCAACUCCUGGGACAAC 3427 GUUGUCCCAGGAGUUGAGGAGGG 3714 6503 6525 AD-1334575 CAGCAACACAGUGACUCCCUC 3428 GAGGGAGUCACUGUGUUGCUGGU 3715 6572 6594 AD-1334576 UGCCCUAGGGACCACCCACAC 3429 GUGUGGGUGGUCCCUAGGGCAGA 3716 6596 6618 AD-1334577 AGUGCCGAACACCAUGGCCAC 3430 GUGGCCAUGGUGUUCGGCACUGG 3717 6623 6645 AD-1334578 AGCCUGGACUUCGGCCACCUC 3431 GAGGUGGCCGAAGUCCAGGCUGU 3718 6692 6714 AD-1334579 ACCCACAUCACAGAGCCUUCC 3432 GGAAGGCUCUGUGAUGUGGGUGG 3719 6729 6751 AD-1334580 GGUGACUUCCCACACCCUAGC 3433 GCUAGGGUGUGGGAAGUCACCGU 3720 6752 6774 AD-1334581 CAACCACCGGUACCACCCAGC 3434 GCUGGGUGGUACCGGUGGUUGCU 3721 6775 6797 AD-1334582 CGACUCCAGCCCUUUCCAGCC 3435 GGCUGGAAAGGGCUGGAGUCGAG 3722 6799 6821 AD-1334583 UAGCAGCAGAACCACCGAGUC 3436 GACUCGGUGGUUCUGCUGCUAGG 3723 6827 6849 AD-1334584 ACCCAGCAAGACCCGCACCUC 3437 GAGGUGCGGGUCUUGCUGGGUGU 3724 6917 6939 AD-1334585 CGGUGGUGACCAUGGGCUGUG 3438 CACAGCCCAUGGUCACCACCGUG 3725 6979 7001 AD-1334586 GUGGCUGGACUACAGCUACCC 3439 GGGUAGCUGUAGUCCAGCCACUC 3726 7022 7044 AD-1334587 UUGACACCUACUCCAACAUCC 3440 GGAUGUUGGAGUAGGUGUCAAAG 3727 7069 7091 AD-1334588 UUGGGCCAGGUCGUGGAAUGC 3441 GCAUUCCACGACCUGGCCCAACU 3728 7173 7195 AD-1334589 CCUGGACUUUGGCCUGGUCUG 3442 CAGACCAGGCCAAAGUCCAGGCU 3729 7196 7218 AD-1334590 AUGUGCUUCAACUAUGAAAUC 3443 GAUUUCAUAGUUGAAGCACAUCU 3730 7248 7270 AD-1334591 UGUGUUCUGCUGCAACUACGG 3444 CCGUAGUUGCAGCAGAACACACG 3731 7271 7293 AD-1334592 CAGCUCUACGGCCAUGCCCUC 3445 GAGGGCAUGGCCGUAGAGCUGGU 3732 7316 7338 AD-1334593 AUCCUCACAGAGCUGACCACA 1848 UGUGGUCAGCUCUGUGAGGAUCC 2172 7359 7381 AD-1334594 CCACUACGACUGAGUCCACUG 3446 CAGUGGACUCAGUCGUAGUGGCU 3733 7384 7406 AD-1334595 GACCACCUGGAUCCUCACAGA 1850 UCUGUGAGGAUCCAGGUGGUCCC 2174 7436 7458 AD-1334596 GAGCACUACAGCCACCGUGAC 3447 GUCACGGUGGCUGUAGUGCUCGG 3734 7460 7482 AD-1334597 CCUCCACCCAGGCAACUGCUG 3448 CAGCAGUUGCCUGGGUGGAGGAG 3735 7510 7532 AD-1334598 GGCCACGACACCCACAGUCAC 3449 GUGACUGUGGGUGUCGUGGCCGU 3736 7553 7575 AD-1334599 GCUCCAAAGCCACUCCCUUCU 179 AGAAGGGAGUGGCUUUGGAGCUG 510 7576 7598 AD-1334559 AGCACUGAGAAGCACAGCCAC 3414 GUGGCUGUGCUUCUCAGUGCUGG 3701 7625 7647 AD-1334600 ACAGCUACCAGCUUUACAGCC 3450 GGCUGUAAAGCUGGUAGCUGUGG 3737 7653 7675 AD-1334562 CCUAUCACAGACCACCACACC 3417 GGUGUGGUGGUCUGUGAUAGGCG 3704 7709 7731 AD-1334563 CCACCAUGUCCACAGCCACAC 3418 GUGUGGCUGUGGACAUGGUGGCC 3705 7735 7757 AD-1334601 UCCUCCACUCCAGAGACUGUC 3451 GACAGUCUCUGGAGUGGAGGAGG 3738 7758 7780 AD-1334602 CCACAGUGCUUACCACCACGG 3452 CCGUGGUGGUAAGCACUGUGGAG 3739 7786 7808 AD-1334603 GCUCACACUACCAAAGUGCUG 3453 CAGCACUUUGGUAGUGUGAGCUG 3740 7863 7885 AD-1334604 UACCACAACCACGGGCUUCAC 3454 GUGAAGCCCGUGGUUGUGGUAGU 3741 7886 7908 AD-1334605 CACGCUUCCAGUGUGGAUCAG 3455 CUGAUCCACACUGGAAGCGUGCG 3742 7940 7962 AD-1334606 ACCCACAACCAGAGGUUCCAC 3456 GUGGAACCUCUGGUUGUGGGUGU 3743 7973 7995 AD-1334607 UGACCACCACCACCACAACUG 3457 CAGUUGUGGUGGUGGUGGUCAGC 3744 8041 8063 AD-1334571 GCCACUGGUUCUAUGGCAACA 1826 UGUUGCCAUAGAACCAGUGGCCA 2150 8064 8086 AD-1334572 CUCCUCUAGCACACAGACCAG 3425 CUGGUCUGUGUGCUAGAGGAGGG 3712 8087 8109 AD-1334573 GGCCACUACGAUCACGGCCAC 3426 GUGGCCGUGAUCGUAGUGGCCGU 3713 8135 8157 AD-1334608 CUCCUCAACUCCAGGGACAAC 3458 GUUGUCCCUGGAGUUGAGGAGGG 3745 8174 8196 AD-1334609 CAGCAGCACAGUGACUCCCUC 3459 GAGGGAGUCACUGUGCUGCUGGU 3746 8243 8265 AD-1334576 UGCCCUAGGGACCACCCACAC 3429 GUGUGGGUGGUCCCUAGGGCAGA 3716 8267 8289 AD-1334610 CACACACGGGCGAUCCCUGUC 3460 GACAGGGAUCGCCCGUGUGUGGU 3747 8315 8337 AD-1334578 AGCCUGGACUUCGGCCACCUC 3431 GAGGUGGCCGAAGUCCAGGCUGU 3718 8363 8385 AD-1334579 ACCCACAUCACAGAGCCUUCC 3432 GGAAGGCUCUGUGAUGUGGGUGG 3719 8400 8422 AD-1334611 CCACCCAGCACUCGACUCCAG 3461 CUGGAGUCGAGUGCUGGGUGGUA 3748 8458 8480 AD-1334612 CAGCCCUCACCCUAGCAGCAG 3462 CUGCUGCUAGGGUGAGGGCUGGA 3749 8486 8508 AD-1334584 ACCCAGCAAGACCCGCACCUC 3437 GAGGUGCGGGUCUUGCUGGGUGU 3724 8588 8610 AD-1334585 CGGUGGUGACCAUGGGCUGUG 3438 CACAGCCCAUGGUCACCACCGUG 3725 8650 8672 AD-1334586 GUGGCUGGACUACAGCUACCC 3439 GGGUAGCUGUAGUCCAGCCACUC 3726 8693 8715 AD-1334587 UUGACACCUACUCCAACAUCC 3440 GGAUGUUGGAGUAGGUGUCAAAG 3727 8740 8762 AD-1334588 UUGGGCCAGGUCGUGGAAUGC 3441 GCAUUCCACGACCUGGCCCAACU 3728 8844 8866 AD-1334589 CCUGGACUUUGGCCUGGUCUG 3442 CAGACCAGGCCAAAGUCCAGGCU 3729 8867 8889 AD-1334590 AUGUGCUUCAACUAUGAAAUC 3443 GAUUUCAUAGUUGAAGCACAUCU 3730 8919 8941 AD-1334591 UGUGUUCUGCUGCAACUACGG 3444 CCGUAGUUGCAGCAGAACACACG 3731 8942 8964 AD-1334613 CUGGAUCCUCACAGAGCAGAC 3463 GUCUGCUCUGUGAGGAUCCAGGU 3750 9026 9048 AD-1334614 CAGCCACUACGACCGCAACCA 1869 UGGUUGCGGUCGUAGUGGCUGCU 2193 9052 9074 AD-1334615 UCCCAAAGUGCUGACCAGCAC 3464 GUGCUGGUCAGCACUUUGGGAGG 3751 9119 9141 AD-1334616 CAGUUCCAAAGCCACUUCCUC 3465 GAGGAAGUGGCUUUGGAACUGGU 3752 9161 9183 AD-1334617 CAAGGACUGCAACCACCCUUC 3466 GAAGGGUGGUUGCAGUCCUUGGA 3753 9190 9212 AD-1334618 GUGCUGACAAGCACAGCCACC 3467 GGUGGCUGUGCUUGUCAGCACUG 3754 9213 9235 AD-1334619 CACAGCUACCAGCUUUACACC 3468 GGUGUAAAGCUGGUAGCUGUGGA 3755 9239 9261 AD-1334620 CUCCUUCACCCUUGGGACCAC 3469 GUGGUCCCAAGGGUGAAGGAGGG 3756 9266 9288 AD-1334621 CCCAGAACAGACCACCACACC 3470 GGUGUGGUGGUCUGUUCUGGGAG 3757 9296 9318 AD-1334622 CACCAUGUCCACAAUCCACCC 3471 GGGUGGAUUGUGGACAUGGUGGC 3758 9323 9345 AD-1334623 CUCCACAGUGCUGACCACGAA 1878 UUCGUGGUCAGCACUGUGGAGGU 2202 9371 9393 AD-1334624 GGCCACCAGUUCCAUGUCCAC 3472 GUGGACAUGGAACUGGUGGCCCU 3759 9407 9429 AD-1334593 AUCCUCACAGAGCUGACCACA 1848 UGUGGUCAGCUCUGUGAGGAUCC 2172 9456 9478 AD-1334625 CCACUACAACUGCAGCCACUG 3473 CAGUGGCUGCAGUUGUAGUGGCU 3760 9481 9503 AD-1334595 GACCACCUGGAUCCUCACAGA 1850 UCUGUGAGGAUCCAGGUGGUCCC 2174 9533 9555 AD-1334626 CAGCACUACAGCCACCGUGAC 3474 GUCACGGUGGCUGUAGUGCUGGG 3761 9557 9579 AD-1334627 ACCCUCAAAGUGCUGACCAGC 3475 GCUGGUCAGCACUUUGAGGGUGC 3762 9630 9652 AD-1334628 ACCCACAGUCAUCAGCUCCAG 3476 CUGGAGCUGAUGACUGUGGGUGU 3763 9662 9684 AD-1334629 CCACUCCCUCCUCCAGUCCAG 3477 CUGGACUGGAGGAGGGAGUGGCU 3764 9685 9707 AD-1334559 AGCACUGAGAAGCACAGCCAC 3414 GUGGCUGUGCUUCUCAGUGCUGG 3701 9725 9747 AD-1334630 CUACCAGCGUUACAGCCAUCC 3478 GGAUGGCUGUAACGCUGGUAGCU 3765 9757 9779 AD-1334562 CCUAUCACAGACCACCACACC 3417 GGUGUGGUGGUCUGUGAUAGGCG 3704 9809 9831 AD-1334563 CCACCAUGUCCACAGCCACAC 3418 GUGUGGCUGUGGACAUGGUGGCC 3705 9835 9857 AD-1334631 CCUCUACUCCAGAGACUGUCC 3479 GGACAGUCUCUGGAGUAGAGGAG 3766 9859 9881 AD-1334602 CCACAGUGCUUACCACCACGA 1857 UCGUGGUGGUAAGCACUGUGGAG 2181 9886 9908 AD-1334632 AGCUCACACUACCAAAGUGCC 3480 GGCACUUUGGUAGUGUGAGCUGU 3767 9962 9984 AD-1334633 CUACCACAACCACGGGCUUCA 1888 UGAAGCCCGUGGUUGUGGUAGUC 2212 9985 10007 AD-1334568 UCCAGUGUGGAUCAGCACAAC 3422 GUUGUGCUGAUCCACACUGGAGG 3709 10046 10068 AD-1334569 ACCCACAACCAGAGGCUCCAC 3423 GUGGAGCCUCUGGUUGUGGGUGU 3710 10073 10095 AD-1334570 CGCCACAGUGCUGACCACCAC 3424 GUGGUGGUCAGCACUGUGGCGGU 3711 10130 10152 AD-1334571 GCCACUGGUUCUAUGGCAACA 1826 UGUUGCCAUAGAACCAGUGGCCA 2150 10164 10186 AD-1334572 CUCCUCUAGCACACAGACCAG 3425 CUGGUCUGUGUGCUAGAGGAGGG 3712 10187 10209 AD-1334634 GGCCACUACGAUCACAGCCAC 3481 GUGGCUGUGAUCGUAGUGGCCGU 3768 10235 10257 AD-1334608 CUCCUCAACUCCAGGGACAAC 3458 GUUGUCCCUGGAGUUGAGGAGGG 3745 10274 10296 AD-1334609 CAGCAGCACAGUGACUCCCUC 3459 GAGGGAGUCACUGUGCUGCUGGU 3746 10343 10365 AD-1334576 UGCCCUAGGGACCACCCACAC 3429 GUGUGGGUGGUCCCUAGGGCAGA 3716 10367 10389 AD-1334635 CACCACGGCCACCACACACGG 3482 CCGUGUGUGGUGGCCGUGGUGUU 3769 10403 10425 AD-1334578 AGCCUGGACUUCGGCCACCUC 3431 GAGGUGGCCGAAGUCCAGGCUGU 3718 10463 10485 AD-1334579 ACCCACAUCACAGAGCCUUCC 3432 GGAAGGCUCUGUGAUGUGGGUGG 3719 10500 10522 AD-1334636 CAGCAGCAACCACCAGUACCA 1891 UGGUACUGGUGGUUGCUGCUGGG 2215 10540 10562 AD-1334612 CAGCCCUCACCCUAGCAGCAG 3462 CUGCUGCUAGGGUGAGGGCUGGA 3749 10586 10608 AD-1334637 CCUCCAGGACCACAGCCACAG 3483 CUGUGGCUGUGGUCCUGGAGGUG 3770 10663 10685 AD-1334584 ACCCAGCAAGACCCGCACCUC 3437 GAGGUGCGGGUCUUGCUGGGUGU 3724 10688 10710 AD-1334638 ACCACGGUGGUGACCACGGGC 3484 GCCCGUGGUCACCACCGUGGUUA 3771 10746 10768 AD-1334586 GUGGCUGGACUACAGCUACCC 3439 GGGUAGCUGUAGUCCAGCCACUC 3726 10793 10815 AD-1334587 UUGACACCUACUCCAACAUCC 3440 GGAUGUUGGAGUAGGUGUCAAAG 3727 10840 10862 AD-1334588 UUGGGCCAGGUCGUGGAAUGC 3441 GCAUUCCACGACCUGGCCCAACU 3728 10944 10966 AD-1334589 CCUGGACUUUGGCCUGGUCUG 3442 CAGACCAGGCCAAAGUCCAGGCU 3729 10967 10989 AD-1334590 AUGUGCUUCAACUAUGAAAUC 3443 GAUUUCAUAGUUGAAGCACAUCU 3730 11019 11041 AD-1334591 UGUGUUCUGCUGCAACUACGG 3444 CCGUAGUUGCAGCAGAACACACG 3731 11042 11064 AD-1334639 CGGCCACGCCCUCCUCAACUC 3485 GAGUUGAGGAGGGCGUGGCCGUA 3772 11095 11117 AD-1334640 CUGGAUCCUCACAAAGCUGAC 3486 GUCAGCUUUGUGAGGAUCCAGGU 3773 11126 11148 AD-1334594 CCACUACGACUGAGUCCACUG 3446 CAGUGGACUCAGUCGUAGUGGCU 3733 11155 11177 AD-1334595 GACCACCUGGAUCCUCACAGA 1850 UCUGUGAGGAUCCAGGUGGUCCC 2174 11207 11229 AD-1334596 GAGCACUACAGCCACCGUGAC 3447 GUCACGGUGGCUGUAGUGCUCGG 3734 11231 11253 AD-1334597 CCUCCACCCAGGCAACUGCUG 3448 CAGCAGUUGCCUGGGUGGAGGAG 3735 11281 11303 AD-1334598 GGCCACGACACCCACAGUCAC 3449 GUGACUGUGGGUGUCGUGGCCGU 3736 11324 11346 AD-1334599 GCUCCAAAGCCACUCCCUUCU 179 AGAAGGGAGUGGCUUUGGAGCUG 510 11347 11369 AD-1334559 AGCACUGAGAAGCACAGCCAC 3414 GUGGCUGUGCUUCUCAGUGCUGG 3701 11396 11418 AD-1334600 ACAGCUACCAGCUUUACAGCC 3450 GGCUGUAAAGCUGGUAGCUGUGG 3737 11424 11446 AD-1334562 CCUAUCACAGACCACCACACC 3417 GGUGUGGUGGUCUGUGAUAGGCG 3704 11480 11502 AD-1334563 CCACCAUGUCCACAGCCACAC 3418 GUGUGGCUGUGGACAUGGUGGCC 3705 11506 11528 AD-1334564 UCCUCCACUCCAGAGACUGCC 3419 GGCAGUCUCUGGAGUGGAGGAGG 3706 11529 11551 AD-1334602 CCACAGUGCUUACCACCACGG 3452 CCGUGGUGGUAAGCACUGUGGAG 3739 11557 11579 AD-1334632 AGCUCACACUACCAAAGUGCC 3480 GGCACUUUGGUAGUGUGAGCUGU 3767 11633 11655 AD-1334633 CUACCACAACCACGGGCUUCA 1888 UGAAGCCCGUGGUUGUGGUAGUC 2212 11656 11678 AD-1334568 UCCAGUGUGGAUCAGCACAAC 3422 GUUGUGCUGAUCCACACUGGAGG 3709 11717 11739 AD-1334641 ACCCACAACCAGUGGCUCCAC 3487 GUGGAGCCACUGGUUGUGGGUGU 3774 11744 11766 AD-1334607 UGACCACCACCACCACAACUG 3457 CAGUUGUGGUGGUGGUGGUCAGC 3744 11812 11834 AD-1334571 GCCACUGGUUCUAUGGCAACA 1826 UGUUGCCAUAGAACCAGUGGCCA 2150 11835 11857 AD-1334572 CUCCUCUAGCACACAGACCAG 3425 CUGGUCUGUGUGCUAGAGGAGGG 3712 11858 11880 AD-1334573 GGCCACUACGAUCACGGCCAC 3426 GUGGCCGUGAUCGUAGUGGCCGU 3713 11906 11928 AD-1334608 CUCCUCAACUCCAGGGACAAC 3458 GUUGUCCCUGGAGUUGAGGAGGG 3745 11945 11967 AD-1334609 CAGCAGCACAGUGACUCCCUC 3459 GAGGGAGUCACUGUGCUGCUGGU 3746 12014 12036 AD-1334576 UGCCCUAGGGACCACCCACAC 3429 GUGUGGGUGGUCCCUAGGGCAGA 3716 12038 12060 AD-1334610 CACACACGGGCGAUCCCUGUC 3460 GACAGGGAUCGCCCGUGUGUGGU 3747 12086 12108 AD-1334578 AGCCUGGACUUCGGCCACCUC 3431 GAGGUGGCCGAAGUCCAGGCUGU 3718 12134 12156 AD-1334579 ACCCACAUCACAGAGCCUUCC 3432 GGAAGGCUCUGUGAUGUGGGUGG 3719 12171 12193 AD-1334611 CCACCCAGCACUCGACUCCAG 3461 CUGGAGUCGAGUGCUGGGUGGUA 3748 12229 12251 AD-1334612 CAGCCCUCACCCUAGCAGCAG 3462 CUGCUGCUAGGGUGAGGGCUGGA 3749 12257 12279 AD-1334584 ACCCAGCAAGACCCGCACCUC 3437 GAGGUGCGGGUCUUGCUGGGUGU 3724 12359 12381 AD-1334638 ACCACGGUGGUGACCACGGGC 3484 GCCCGUGGUCACCACCGUGGUUA 3771 12417 12439 AD-1334586 GUGGCUGGACUACAGCUACCC 3439 GGGUAGCUGUAGUCCAGCCACUC 3726 12464 12486 AD-1334587 UUGACACCUACUCCAACAUCC 3440 GGAUGUUGGAGUAGGUGUCAAAG 3727 12511 12533 AD-1334588 UUGGGCCAGGUCGUGGAAUGC 3441 GCAUUCCACGACCUGGCCCAACU 3728 12615 12637 AD-1334589 CCUGGACUUUGGCCUGGUCUG 3442 CAGACCAGGCCAAAGUCCAGGCU 3729 12638 12660 AD-1334590 AUGUGCUUCAACUAUGAAAUC 3443 GAUUUCAUAGUUGAAGCACAUCU 3730 12690 12712 AD-1334591 UGUGUUCUGCUGCAACUACGG 3444 CCGUAGUUGCAGCAGAACACACG 3731 12713 12735 AD-1334592 CAGCUCUACGGCCAUGCCCUC 3445 GAGGGCAUGGCCGUAGAGCUGGU 3732 12758 12780 AD-1334642 GACGACCUGGAUCCUCACAGA 1897 UCUGUGAGGAUCCAGGUCGUCCC 2221 12791 12813 AD-1334643 UGACCACAACAGCCACUACGA 1898 UCGUAGUGGCUGUUGUGGUCAGC 2222 12814 12836 AD-1334644 AUCCACUGGAUCCACGGCCAC 3488 GUGGCCGUGGAUCCAGUGGAUGC 3775 12839 12861 AD-1334645 CUCCCAAAGUGCUGACCAGCC 3489 GGCUGGUCAGCACUUUGGGAGGG 3776 12889 12911 AD-1334616 CAGUUCCAAAGCCACUUCCUC 3465 GAGGAAGUGGCUUUGGAACUGGU 3752 12932 12954 AD-1334617 CAAGGACUGCAACCACCCUUC 3466 GAAGGGUGGUUGCAGUCCUUGGA 3753 12961 12983 AD-1334646 CCACCAAAUCCACAGCUACCA 1901 UGGUAGCUGUGGAUUUGGUGGCU 2225 13000 13022 AD-1334621 CCCAGAACAGACCACCACACC 3470 GGUGUGGUGGUCUGUUCUGGGAG 3757 13067 13089 AD-1334622 CACCAUGUCCACAAUCCACCC 3471 GGGUGGAUUGUGGACAUGGUGGC 3758 13094 13116 AD-1334623 CUCCACAGUGCUGACCACGAA 1878 UUCGUGGUCAGCACUGUGGAGGU 2202 13142 13164 AD-1334647 GGCCACCAGUUCCACGUCCAC 3490 GUGGACGUGGAACUGGUGGCCCU 3777 13178 13200 AD-1334593 AUCCUCACAGAGCUGACCACA 1848 UGUGGUCAGCUCUGUGAGGAUCC 2172 13227 13249 AD-1334625 CCACUACAACUGCAGCCACUG 3473 CAGUGGCUGCAGUUGUAGUGGCU 3760 13252 13274 AD-1334595 GACCACCUGGAUCCUCACAGA 1850 UCUGUGAGGAUCCAGGUGGUCCC 2174 13304 13326 AD-1334643 UGACCACAACAGCCACUACGA 1898 UCGUAGUGGCUGUUGUGGUCAGC 2222 13327 13349 AD-1334595 GACCACCUGGAUCCUCACAGA 1850 UCUGUGAGGAUCCAGGUGGUCCC 2174 13391 13413 AD-1334596 GAGCACUACAGCCACCGUGAC 3447 GUCACGGUGGCUGUAGUGCUCGG 3734 13415 13437 AD-1334597 CCUCCACCCAGGCAACUGCUG 3448 CAGCAGUUGCCUGGGUGGAGGAG 3735 13465 13487 AD-1334648 UGAGCACCACGGCCACGACAC 3491 GUGUCGUGGCCGUGGUGCUCACA 3778 13498 13520 AD-1334557 CAGCUCCAAAGCCACUCCCUC 3412 GAGGGAGUGGCUUUGGAGCUGGU 3699 13529 13551 AD-1334649 CCAGGGACUGCAACUGCCCUU 3492 AAGGGCAGUUGCAGUCCCUGGAC 3779 13557 13579 AD-1334559 AGCACUGAGAAGCACAGCCAC 3414 GUGGCUGUGCUUCUCAGUGCUGG 3701 13580 13602 AD-1334600 ACAGCUACCAGCUUUACAGCC 3450 GGCUGUAAAGCUGGUAGCUGUGG 3737 13608 13630 AD-1334562 CCUAUCACAGACCACCACACC 3417 GGUGUGGUGGUCUGUGAUAGGCG 3704 13664 13686 AD-1334563 CCACCAUGUCCACAGCCACAC 3418 GUGUGGCUGUGGACAUGGUGGCC 3705 13690 13712 AD-1334601 UCCUCCACUCCAGAGACUGUC 3451 GACAGUCUCUGGAGUGGAGGAGG 3738 13713 13735 AD-1334565 CUCCACAGUGCUUACCGCCAC 3420 GUGGCGGUAAGCACUGUGGAGGU 3707 13739 13761 AD-1334632 AGCUCACACUACCAAAGUGCC 3480 GGCACUUUGGUAGUGUGAGCUGU 3767 13817 13839 AD-1334633 CUACCACAACCACGGGCUUCA 1888 UGAAGCCCGUGGUUGUGGUAGUC 2212 13840 13862 AD-1334568 UCCAGUGUGGAUCAGCACAAC 3422 GUUGUGCUGAUCCACACUGGAGG 3709 13901 13923 AD-1334641 ACCCACAACCAGUGGCUCCAC 3487 GUGGAGCCACUGGUUGUGGGUGU 3774 13943 13965 AD-1334650 CGCCAGAGUGCUGACCACCAC 3493 GUGGUGGUCAGCACUCUGGCGGU 3780 14000 14022 AD-1334571 GCCACUGGUUCUAUGGCAACA 1826 UGUUGCCAUAGAACCAGUGGCCA 2150 14034 14056 AD-1334572 CUCCUCUAGCACACAGACCAG 3425 CUGGUCUGUGUGCUAGAGGAGGG 3712 14057 14079 AD-1334573 GGCCACUACGAUCACGGCCAC 3426 GUGGCCGUGAUCGUAGUGGCCGU 3713 14105 14127 AD-1334651 CAGGGACAACACCCAUCACCC 3494 GGGUGAUGGGUGUUGUCCCUGGA 3781 14155 14177 AD-1334652 CUCCAAAGCCACUUCCUCCUC 3495 GAGGAGGAAGUGGCUUUGGAGCU 3782 14216 14238 AD-1334617 CAAGGACUGCAACCACCCUUC 3466 GAAGGGUGGUUGCAGUCCUUGGA 3753 14242 14264 AD-1334653 UGCUGACAAGCACAGCCACAA 1908 UUGUGGCUGUGCUUGUCAGCACU 2232 14266 14288 AD-1334619 CACAGCUACCAGCUUUACACC 3468 GGUGUAAAGCUGGUAGCUGUGGA 3755 14291 14313 AD-1334654 CUCCACCCUGUGGACCACGUG 3496 CACGUGGUCCACAGGGUGGAGGA 3783 14321 14343 AD-1334655 CCCAGCACAGACCACCACACC 3497 GGUGUGGUGGUCUGUGCUGGGAC 3784 14348 14370 AD-1334656 GUCCACCAUGUCCACAAUCCA 1911 UGGAUUGUGGACAUGGUGGACAU 2235 14372 14394 AD-1334657 CUCCUCUACUCCAGAGACCAC 3498 GUGGUCUCUGGAGUAGAGGAGGU 3785 14396 14418 AD-1334658 CUCCACAGUGCUGACCACCAC 3499 GUGGUGGUCAGCACUGUGGAGGU 3786 14423 14445 AD-1334659 GGCCACCAAUUCCACGGCCAC 3500 GUGGCCGUGGAAUUGGUGGCCCU 3787 14459 14481 AD-1334660 UGACCACAACAGCCACUACAA 1915 UUGUAGUGGCUGUUGUGGUCAGC 2239 14521 14543 AD-1334661 UGGAUCCACGGCCACCCUGUC 3501 GACAGGGUGGCCGUGGAUCCAGU 3788 14552 14574 AD-1334595 GACCACCUGGAUCCUCACAGA 1850 UCUGUGAGGAUCCAGGUGGUCCC 2174 14585 14607 AD-1334662 GAGCACUAUAGCCACCGUGAU 3502 AUCACGGUGGCUAUAGUGCUCGG 3789 14609 14631 AD-1334663 CCACUCUGGGAACAGCUCACA 1918 UGUGAGCUGUUCCCAGAGUGGAG 2242 14662 14684 AD-1334664 CAUGGCCACUAUGCCCACAGC 3503 GCUGUGGGCAUAGUGGCCAUGGU 3790 14702 14724 AD-1334665 UGCCUCCACGGUUCCCAGCUC 3504 GAGCUGGGAACCGUGGAGGCAGU 3791 14726 14748 AD-1334666 GCCAACCUUCAGCGUGUCCAC 3505 GUGGACACGCUGAAGGUUGGCAG 3792 14795 14817 AD-1334667 UCCUCCUCAGUCCUCACCACC 3506 GGUGGUGAGGACUGAGGAGGACA 3793 14820 14842 AD-1334668 UCCCACUUCUCUACUCCCUGC 3507 GCAGGGAGUAGAGAAGUGGGAGC 3794 14865 14887 AD-1334669 GCAUUUGGACAGUUUUUCUCG 3508 CGAGAAAAACUGUCCAAAUGCCC 3795 14895 14917 AD-1334670 GAAGUCAUCUACAAUAAGACC 3509 GGUCUUAUUGUAGAUGACUUCCC 3796 14922 14944 AD-1334671 CUGCCAUUUCUACGCAGUGUG 3510 CACACUGCGUAGAAAUGGCAGCC 3797 14954 14976 AD-1334672 CACUGUGACAUUGACCGCUUC 3511 GAAGCGGUCAAUGUCACAGUGCU 3798 14982 15004 AD-1334673 UGUGACAAUGCCAUCCCUCUC 3512 GAGAGGGAUGGCAUUGUCACAGC 3799 15075 15097 AD-1334674 ACCCUGGAGAACUGCACGGUG 3513 CACCGUGCAGUUCUCCAGGGUCC 3800 15117 15139 AD-1334675 GUGGGUGACAACCGUGUCGUC 3514 GACGACACGGUUGUCACCCACGC 3801 15147 15169 AD-1334676 GACCCAAAGCCUGUGGCCAAC 3515 GUUGGCCACAGGCUUUGGGUCCA 3802 15174 15196 AD-1334677 CUGCGUGAACAAGCACCUGCC 3516 GGCAGGUGCUUGUUCACGCAGGU 3803 15200 15222 AD-1334678 UCAAAGUGUCGGACCCGAGCC 3517 GGCUCGGGUCCGACACUUUGAUG 3804 15223 15245 AD-1334679 CCUGUGACUUCCACUAUGAGU 3518 ACUCAUAGUGGAAGUCACAGGGC 3805 15247 15269 AD-1334680 GAGUGCAUCUGCAGCAUGUGG 3519 CCACAUGCUGCAGAUGCACUCGC 3806 15270 15292 AD-1334681 CCCACUAUUCCACCUUUGACG 3520 CGUCAAAGGUGGAAUAGUGGGAG 3807 15298 15320 AD-1334682 ACCUAUGUCCUCAUGAGAGAG 3521 CUCUCUCAUGAGGACAUAGGUGC 3808 15348 15370 AD-1334683 CACGCUUUGGGAAUCUCAGCC 3522 GGCUGAGAUUCCCAAAGCGUGCA 3809 15376 15398 AD-1334684 CUGGACAACCACUACUGCACG 3523 CGUGCAGUAGUGGUUGUCCAGGU 3810 15402 15424 AD-1334685 CUCAGCAUCCACUACAAGUCC 3524 GGACUUGUAGUGGAUGCUGAGGG 3811 15462 15484 AD-1334686 GUCCUCACUGUCACCAUGGUG 3525 CACCAUGGUGACAGUGAGGACGA 3812 15492 15514 AD-1334687 CCUGAUCCUGUUUGACCAAAU 3526 AUUUGGUCAAACAGGAUCAGGCC 3813 15530 15552 AD-1334688 AGCGGUUUCAGCAAGAACGGC 3527 GCCGUUCUUGCUGAAACCGCUGC 3814 15561 15583 AD-1334689 CGUGUGGACAUUCCUGCCCUG 3528 CAGGGCAGGAAUGUCCACACGCA 3815 15615 15637 AD-1334690 GUGAGCGUCACCUUCAAUGGC 3529 GCCAUUGAAGGUGACGCUCACGC 3816 15639 15661 AD-1334691 AGCCUCUUCCACAACAACACC 3530 GGUGUUGUUGUGGAAGAGGCUGU 3817 15687 15709 AD-1334692 UGCACCAACAACCAGAGGGAC 3531 GUCCCUCUGGUUGUUGGUGCAGG 3818 15726 15748 AD-1334693 UGUCUCCAGCGGGACGGAACC 3532 GGUUCCGUCCCGCUGGAGACAGU 3819 15750 15772 AD-1334694 CGCCAGUUGCAAGGACAUGGC 3533 GCCAUGUCCUUGCAACUGGCGGC 3820 15776 15798 AD-1334695 CGACAGCAGAAAGGAUGGCUG 3534 CAGCCAUCCUUUCUGCUGUCGGG 3821 15815 15837 AD-1334696 CCGCUCUGUGAUCUGAUGCUG 3535 CAGCAUCAGAUCACAGAGCGGCU 3822 15921 15943 AD-1334697 CAGGUCUUUGCUGAGUGCCAC 3536 GUGGCACUCAGCAAAGACCUGGC 3823 15945 15967 AD-1334698 GGGCCCAUUCUUCAACGCCUG 3537 CAGGCGUUGAAGAAUGGGCCCGG 3824 15980 16002 AD-1334699 GAGGCUUACGCAGAGCUCUGC 3538 GCAGAGCUCUGCGUAAGCCUCCA 3825 16050 16072 AD-1334700 AGUGUGCAGUGACUGGCGAGG 3539 CCUCGCCAGUCACUGCACACUCC 3826 16082 16104 AD-1334701 CCACCAAAGUGUACAAGCCAU 3540 AUGGCUUGUACACUUUGGUGGGU 3827 16138 16160 AD-1334702 CUGCAACUCUAGGAACCAGAG 3541 CUCUGGUUCCUAGAGUUGCAGGU 3828 16181 16203 AD-1334703 CAGAUCCUCUUCAACGCACAC 3542 GUGUGCGUUGAAGAGGAUCUGGU 3829 16248 16270 AD-1334704 GGGCAUCUGCGUGCAGGCCUG 3543 CAGGCCUGCACGCAGAUGCCCAU 3830 16271 16293 AD-1334705 CGAUGGGUUUCCUAAAUUUCC 3544 GGAAAUUUAGGAAACCCAUCGGG 3831 16307 16329 AD-1334706 GGUCAGCAACUGCCAGUCCUG 3545 CAGGACUGGCAGUUGCUGACCCA 3832 16340 16362 AD-1334707 GAGGGUUCAGUGUCGGUGCAG 3546 CUGCACCGACACUGAACCCUCGU 3833 16371 16393 AD-1334708 CGGCUUCGUAACCGUGACCAG 3547 CUGGUCACGGUUACGAAGCCGGG 3834 16445 16467 AD-1334709 CGUGUGCAACACAACCACCUG 3548 CAGGUGGUUGUGUUGCACACGCA 3835 16505 16527 AD-1334710 CAGGAGUCCAUCUGCACCCAG 3549 CUGGGUGCAGAUGGACUCCUGCC 3836 16557 16579 AD-1334711 CUGUCCCACCUUCCGCUGCAG 3550 CUGCAGCGGAAGGUGGGACAGCA 3837 16592 16614 AD-1334712 UCAGCUGUGUUCGUACAAUGG 3551 CCAUUGUACGAACACAGCUGAGG 3838 16616 16638 AD-1334713 UUGGUGCAACCUUCCCAGGCG 3552 CGCCUGGGAAGGUUGCACCAACC 3839 16651 16673 AD-1334714 UCCCUGCCACAUGUGUACCUG 3553 CAGGUACACAUGUGGCAGGGAAG 3840 16676 16698 AD-1334715 ACGGUGCAAUGUCAGGAGGAU 3554 AUCCUCCUGACAUUGCACCGUUG 3841 16722 16744 AD-1334716 CUGCAACAAUACUACCUGUCC 3555 GGACAGGUAGUAUUGUUGCAGGC 3842 16745 16767 AD-1334717 GGGCUUUGAGUACAAGAGAGU 3556 ACUCUCUUGUACUCAAAGCCCUG 3843 16769 16791 AD-1334718 GUCCAGCUGAAUGAAACCUGG 3557 CCAGGUUUCAUUCAGCUGGACUG 3844 16851 16873 AD-1334719 CAACAGCCAUGUGGACAACUG 3558 CAGUUGUCCACAUGGCUGUUGAC 3845 16874 16896 AD-1334720 CCGUGUACCUCUGUGAGGCUG 3559 CAGCCUCACAGAGGUACACGGUG 3846 16897 16919 AD-1334721 GGUGGAGUCCAUUUGCUGACC 3560 GGUCAGCAAAUGGACUCCACCCU 3847 16920 16942 AD-1334722 CUGCCCAGAUGUGUCCAGCUG 3561 CAGCUGGACACAUCUGGGCAGGA 3848 16955 16977 AD-1334723 GCUGCUACUCCUGUGAGGAGG 3562 CCUCCUCACAGGAGUAGCAGCAG 3849 17002 17024 AD-1334724 UCCUGUCAAGUCCGCAUCAAC 3563 GUUGAUGCGGACUUGACAGGAGU 3850 17025 17047 AD-1334725 GACCAUCCUGUGGCACCAGGG 3564 CCCUGGUGCCACAGGAUGGUCGU 3851 17048 17070 AD-1320631 GGUCAACAUCACCUUCUGCGA 1981 UCGCAGAAGGUGAUGUUGACCUC 2305 17081 17103 AD-1334726 CGUCCAAGUACUCAGCAGAGG 3565 CCUCUGCUGAGUACUUGGACGCU 3852 17119 17141 AD-1334727 AUGCAGCACCAGUGCACCUGC 3566 GCAGGUGCACUGGUGCUGCAUGG 3853 17148 17170 AD-1334728 GCCCUUGCACUGUCCUAACGG 3567 CCGUUAGGACAGUGCAAGGGCAC 3854 17201 17223 AD-1334729 CUGCACACCUACACCCACGUG 3568 CACGUGGGUGUAGGUGUGCAGGA 3855 17232 17254 AD-1334730 GCACGCCCUUCUGUGUCCCUG 3569 CAGGGACACAGAAGGGCGUGCAG 3856 17266 17288 AD-1334731 ACUGCUGUCUGAGAACGUUCU 336 AGAACGUUCUCAGACAGCAGUGG 668 17334 17356 AD-1334732 CAUGCUCUGUCCACCUGGAGC 3570 GCUCCAGGUGGACAGAGCAUGGG 3857 17366 17388 AD-1334733 GCAUUGUCUGAUCAUGAAAAC 3571 GUUUUCAUGAUCAGACAAUGCAC 3858 17395 17417 AD-1334734 GGCGCCACUCAGGAGUCCUAC 3572 GUAGGACUCCUGAGUGGCGCCCU 3859 17542 17564 AD-1334735 CUCCCUGAUGUCACUGGGACG 3573 CGUCCCAGUGACAUCAGGGAGGG 3860 17598 17620 AD-1334736 CUGGAACAAACUAAGCAUGUG 3574 CACAUGCUUAGUUUGUUCCAGGG 3861 17621 17643 AD-1334737 GCACGGAUUCCAGCUGGCCAC 3575 GUGGCCAGCUGGAAUCCGUGCUG 3862 17682 17704 AD-1334738 GACAGGCUGGUCCAGGCAAGG 3576 CCUUGCCUGGACCAGCCUGUCUG 3863 17720 17742 AD-1334739 GCUGCCAGGAAGCUGCGACAG 3577 CUGUCGCAGCUUCCUGGCAGCAG 3864 17747 17769 AD-1334740 GCAGGGUAACUCAGGGCUGAG 3578 CUCAGCCCUGAGUUACCCUGCAG 3865 17796 17818 AD-1334741 GCAACGGCCAGGUCAGAGAGG 3579 CCUCUCUGACCUGGCCGUUGCGA 3866 17820 17842 AD-1334742 AGCCCAGUUUUGCAAAUAAAC 3580 GUUUAUUUGCAAAACUGGGCUGG 3867 17874 17896

TABLE 7 Modified Sense and Antisense Strand MUC5B dsRNA Sequences SEQ SEQ mRNA Target SEQ Duplex Sense Sequence ID Antisense Sequence ID Sequence ID Name 5′ to 3′ NO: 5′ to 3′ NO: 5′ to 3′ NO: AD-1334421 ususggc(Uhd)CfuGfG 3868 VPusAfsgcaUfgGfCfc 2647 TGTTGGCTCTGGCGGCCA 2971 fCfggccaugcsusa gccAfgAfgccaascsa TGCTC AD-1334422 csusggg(Ahd)GfaAfU 3869 VPusUfsgugCfcCfUfg 2648 AGCTGGGAGAATGCAGGG 2972 fGfcagggcacsasa cauUfcUfcccagscsu CACAC AD-1334423 gscsgcg(Uhd)GfaGfC 3870 VPusGfsuggAfaCfAfa 2649 CGGCGCGTGAGCTTTGTT 2973 fUfuuguuccascsa agcUfcAfcgcgcscsg CCACC AD-1334424 usgsggc(Ghd)GfgUfG 3871 VPusAfsgguGfcUfGfc 2650 AATGGGCGGGTGTGCAGC 2974 fUfgcagcaccsusa acaCfcCfgcccasusu ACCTG AD-1334425 cscsacu(Ahd)CfaAfG 3872 VPusCfsgucGfaAfGfg 2651 TTCCACTACAAGACCTTC 2975 fAfccuucgacsgsa ucuUfgUfaguggsasa GACGG AD-1334426 cscsuuu(Ghd)CfaAfC 3873 VPusAfsgaaCfaCfGfu 2652 GGCCTTTGCAACTACGTG 2976 fUfacguguucsusa aguUfgCfaaaggscsc TTCTC AD-1334427 gsasgga(Chd)UfuCfA 3874 VPusAfsgcuGfgAfCfg 2653 ACGAGGACTTCAACGTCC 2977 fAfcguccagcsusa uugAfaGfuccucsgsu AGCTA AD-1334428 ascsccg(Uhd)GfuUfG 3875 VPusGfsccuUfgAfUfg 2654 TCACCCGTGTTGTCATCA 2978 fUfcaucaaggscsa acaAfcAfcgggusgsa AGGCC AD-1334429 gsgscuc(Chd)GfuCfC 3876 VPusCfscauUfgAfUfg 2655 ACGGCTCCGTCCTCATCA 2979 fUfcaucaaugsgsa aggAfcGfgagccsgsu ATGGG AD-1334430 gscsugc(Chd)UfuAfC 3877 VPusCfsaguGfcGfGfc 2656 GAGCTGCCTTACAGCCGC 2980 fAfgccgcacusgsa uguAfaGfgcagcsusc ACTGG AD-1334431 gsascua(Chd)AfuCfA 3878 VPusAfsugcUfgAfCfc 2657 GGGACTACATCAAGGTCA 2981 fAfggucagcasusa uugAfuGfuagucscsc GCATC AD-1334432 gscsuga(Chd)AfuUfC 3879 VPusCfsguuCfcAfCfa 2658 GTGCTGACATTCCTGTGG 2982 fCfuguggaacsgsa ggaAfuGfucagcsasc AACGG AD-1334433 csusgga(Uhd)CfcCfA 3880 VPusUfsuggCfgUfAfu 2659 AGCTGGATCCCAAATACG 2983 fAfauacgccasasa uugGfgAfuccagscsu CCAAC AD-1334434 gscscuu(Chd)AfaCfG 3881 VPusGfscauAfgAfAfc 2660 CGGCCTTCAACGAGTTCT 2984 fAfguucuaugscsa ucgUfuGfaaggcscsg ATGCC AD-1334435 asasccu(Ghd)CfaGfA 3882 VPusCfscauCfcAfAfc 2661 GGAACCTGCAGAAGTTGG 2985 fAfguuggaugsgsa uucUfgCfagguuscsc ATGGG AD-1334436 usgscac(Ghd)GfaCfG 3883 VPusAfsugcCfcUfCfc 2662 ACTGCACGGACGAGGAGG 2986 fAfggagggcasusa ucgUfcCfgugcasgsu GCATC AD-1334437 ususugc(Ghd)GfaGfU 3884 VPusAfsgugCfgUfGfg 2663 CCTTTGCGGAGTGCCACG 2987 fGfccacgcacsusa cacUfcCfgcaaasgsg CACTG AD-1334438 gsascag(Chd)AfcUfG 3885 VPusGfsccaGfgUfAfc 2664 TGGACAGCACTGCGTACC 2988 fCfguaccuggscsa gcaGfuGfcugucscsa TGGCC AD-1334439 gscscac(Chd)UfuUfG 3886 VPusGfsaguAfuUfCfc 2665 GTGCCACCTTTGTGGAAT 2989 fUfggaauacuscsa acaAfaGfguggcsasc ACTCA AD-1334440 csusgga(Ghd)GfuGfC 3887 VPusAfsgagCfuCfAfg 2666 AACTGGAGGTGCCCTGAG 2990 fCfcugagcucsusa ggcAfcCfuccagsusu CTCTG AD-1334441 csuscaa(Chd)AfuGfC 3888 VPusUfsccuGfgUfGfc 2667 CCCTCAACATGCAGCACC 2991 fAfgcaccaggsasa ugcAfuGfuugagsgsg AGGAG AD-1334442 ascsccu(Ghd)CfaCfG 3889 VPusAfsgcaGfgUfGfu 2668 TCACCCTGCACGGACACC 2992 fGfacaccugcsusa ccgUfgCfagggusgsa TGCTC AD-1334443 gsgsacc(Ahd)CfuGfU 3890 VPusAfsgccGfuCfCfa 2669 GAGGACCACTGTGTGGAC 2993 fGfuggacggcsusa cacAfgUfgguccsusc GGCTG AD-1334444 gscsugg(Ahd)UfgAfC 3891 VPusAfsgugCfgUfGfa 2670 GTGCTGGATGACATCACG 2994 fAfucacgcacsusa uguCfaUfccagcsasc CACTC AD-1334445 csusccu(Uhd)CfaAfC 3892 VPusUfsgcaGfgUfGfg 2671 ACCTCCTTCAACACCACC 2995 fAfccaccugcsasa uguUfgAfaggagsgsu TGCAG AD-1334446 csusaug(Ghd)CfaGfU 3893 VPusAfsgguCfcUfGfg 2672 GGCTATGGCAGTGCCAGG 2996 fGfccaggaccsusa cacUfgCfcauagscsc ACCTG AD-1334447 ascscua(Uhd)GfaUfG 3894 VPusUfsagaGfuUfUfc 2673 CCACCTATGATGAGAAAC 2997 fAfgaaacucusasa ucaUfcAfuaggusgsg TCTAC AD-1334448 asgscua(Chd)GfuUfC 3895 VPusUfsucuUfgGfAfc 2674 GCAGCTACGTTCTGTCCA 2998 fUfguccaagasasa agaAfcGfuagcusgsc AGAAA AD-1334449 gsascag(Chd)AfgCfU 3896 VPusAfsgcaCfgGfUfg 2675 CCGACAGCAGCTTCACCG 2999 fUfcaccgugcsusa aagCfuGfcugucsgsg TGCTG AD-1334450 gsgsaca(Ahd)CfgAfG 3897 VPusUfscagGfcAfGfu 2676 ACGGACAACGAGAACTGC 3000 fAfacugccugsasa ucuCfgUfuguccsgsu CTGAA AD-1334451 uscscuc(Ahd)AfcUfC 3898 VPusCfsgugUfaGfAfu 2677 GTTCCTCAACTCCATCTA 3001 fCfaucuacacsgsa ggaGfuUfgaggasasc CACGC AD-1334452 gscscaa(Chd)AfuCfA 3899 VPusGfsugaAfcAfGfg 2678 CAGCCAACATCACCCTGT 3002 fCfccuguucascsa gugAfuGfuuggcsusg TCACA AD-1334453 uscsgag(Chd)UfuCfU 3900 VPusAfsccaCfgAfUfg 2679 CCTCGAGCTTCTTCATCG 3003 fUfcaucguggsusa aagAfaGfcucgasgsg TGGTG AD-1334454 gscscac(Uhd)CfaUfG 3901 VPusCfsaaaCfaCfCfu 2680 GTGCCACTCATGCAGGTG 3004 fCfagguguuusgsa gcaUfgAfguggcsasc TTTGT AD-1334455 gsgsgaa(Chd)UfuCfA 3902 VPusUfsgguUfcUfGfg 2681 GTGGGAACTTCAACCAGA 3005 fAfccagaaccsasa uugAfaGfuucccsasc ACCAG AD-1334456 usgsacg(Ahd)CfuUfC 3903 VPusUfsgagGfgCfCfg 2682 GCTGACGACTTCACGGCC 3006 fAfcggcccucsasa ugaAfgUfcgucasgsc CTCAG AD-1334457 asgsccu(Uhd)CfgCfC 3904 VPusUfsccaGfgUfGfu 2683 GCAGCCTTCGCCAACACC 3007 fAfacaccuggsasa uggCfgAfaggcusgsc TGGAA AD-1334458 cscsagg(Ahd)AfcAfG 3905 VPusGfsuccUfcAfAfa 2684 TGCCAGGAACAGCTTTGA 3008 fCfuuugaggascsa gcuGfuUfccuggscsa GGACC AD-1334459 gsusgga(Ghd)AfaUfG 3906 VPusGfscguAfgUfUfc 2685 GTGTGGAGAATGAGAACT 3009 fAfgaacuacgscsa ucaUfuCfuccacsasc ACGCC AD-1334460 csasaca(Ghd)UfgCfC 3907 VPusAfsgcgCfgAfGfa 2686 CCCAACAGTGCCTTCTCG 3010 fUfucucgcgcsusa aggCfaCfuguugsgsg CGCTG AD-1334461 csusucc(Ahd)CfuCfG 3908 VPusAfscauGfcAfGfu 2687 CCCTTCCACTCGAACTGC 3011 fAfacugcaugsusa ucgAfgUfggaagsgsg ATGTT AD-1334462 csasccu(Ghd)CfaAfC 3909 VPusUfsccgCfuCfAfc 2688 GACACCTGCAACTGTGAG 3012 fUfgugagcggsasa aguUfgCfaggugsusc CGGAG AD-1334463 cscsucc(Uhd)AfuGfU 3910 VPusAfscagGfcGfUfg 2689 GTCCTCCTATGTGCACGC 3013 fGfcacgccugsusa cacAfuAfggaggsasc CTGTG AD-1334464 gsgscgu(Ahd)CfaGfC 3911 VPusCfsaguCfgCfUfg 2690 AGGGCGTACAGCTCAGCG 3014 fUfcagcgacusgsa agcUfgUfacgccscsu ACTGG AD-1334465 ascscaa(Ghd)UfaCfA 3912 VPusCfsaguUfcUfGfc 2691 GCACCAAGTACATGCAGA 3015 fUfgcagaacusgsa augUfaCfuuggusgsc ACTGC AD-1334466 usascgc(Chd)UfaCfG 3913 VPusGfscauCfcAfCfc 2692 GCTACGCCTACGTGGTGG 3016 fUfgguggaugscsa acgUfaGfgcguasgsc ATGCC AD-1334467 gscsagc(Ghd)UfuUfC 3914 VPusAfsggcAfcGfAfa 2693 CTGCAGCGTTTCCTTCGT 3017 fCfuucgugccsusa ggaAfaCfgcugcsasg GCCTG AD-1334468 gscsacc(Uhd)UfcCfU 3915 VPusCfsgcgUfcAfUfu 2694 GGGCACCTTCCTCAATGA 3018 fCfaaugacgcsgsa gagGfaAfggugcscsc CGCGG AD-1334469 csusgga(Ghd)AfgGfU 3916 VPusGfsucgUfgCfAfc 2695 TCCTGGAGAGGTGGTGCA 3019 fGfgugcacgascsa cacCfuCfuccagsgsa CGACG AD-1334470 cscsgug(Uhd)GfuUfC 3917 VPusAfscccGfuAfCfa 2696 CGCCGTGTGTTCATGTAC 3020 fAfuguacgggsusa ugaAfcAfcacggscsg GGGTG AD-1334471 cscsucu(Chd)UfgCfA 3918 VPusUfsgugCfuUfUfu 2697 AGCCTCTCTGCAGAAAAG 3021 fGfaaaagcacsasa cugCfaGfagaggscsu CACAG AD-1334472 gsgsacu(Ghd)CfaGfC 3919 VPusCfscgaGfcUfGfu 2698 CTGGACTGCAGCAACAGC 3022 fAfacagcucgsgsa ugcUfgCfaguccsasg TCGGC AD-1334473 csusguu(Uhd)CfaGfC 3920 VPusCfsgcaGfuGfUfg 2699 GGCTGTTTCAGCACACAC 3023 fAfcacacugcsgsa ugcUfgAfaacagscsc TGCGT AD-1334474 csusgca(Uhd)UfgCfC 3921 VPusAfsgucCfuCfCfu 2700 GGCTGCATTGCCGAGGAG 3024 fGfaggaggacsusa cggCfaAfugcagscsc GACTG AD-1334475 csasccu(Ahd)CfaAfG 3922 VPusUfscucUfcCfAfg 2701 GCCACCTACAAGCCTGGA 3025 fCfcuggagagsasa gcuUfgUfaggugsgsc GAGAC AD-1334476 csgsacu(Ghd)CfaAfC 3923 VPusAfsgguGfcAfGfg 2702 GTCGACTGCAACACCTGC 3026 fAfccugcaccsusa uguUfgCfagucgsasc ACCTG AD-1334477 gsasacc(Ghd)GfaGfG 3924 VPusUfsgcaCfuCfCfc 2703 AGGAACCGGAGGTGGGAG 3027 fUfgggagugcsasa accUfcCfgguucscsu TGCAG AD-1334478 usgsgcc(Ahd)CfuUfC 3925 VPusCfsaaaGfgUfGfa 2704 GATGGCCACTTCATCACC 3028 fAfucaccuuusgsa ugaAfgUfggccasusc TTTGA AD-1334479 csgsauc(Ghd)CfuAfC 3926 VPusCfsuucAfaAfGfc 2705 GGCGATCGCTACAGCTTT 3029 fAfgcuuugaasgsa uguAfgCfgaucgscsc GAAGG AD-1334480 gscsugc(Ghd)AfgUfA 3927 VPusGfsgccAfaGfAfu 2706 CAGCTGCGAGTACATCTT 3030 fCfaucuuggcscsa guaCfuCfgcagcsusg GGCCC AD-1334481 csusucc(Ghd)CfaUfC 3928 VPusUfscucGfgUfGfa 2707 ACCTTCCGCATCGTCACC 3031 fGfucaccgagsasa cgaUfgCfggaagsgsu GAGAA AD-1334482 gscscau(Chd)AfaGfC 3929 VPusUfsccaCfgAfAfg 2708 AGGCCATCAAGCTCTTCG 3032 fUfcuucguggsasa agcUfuGfauggcscsu TGGAG AD-1334483 usascga(Ghd)CfuGfA 3930 VPusUfscuuGfgAfGfg 2709 GCTACGAGCTGATCCTCC 3033 fUfccuccaagsasa aucAfgCfucguasgsc AAGAG AD-1334484 gsasccu(Uhd)UfaAfG 3931 VPusUfscgcCfaCfCfg 2710 GGGACCTTTAAGGCGGTG 3034 fGfcgguggcgsasa ccuUfaAfaggucscsc GCGAG AD-1334485 ascsccu(Ahd)CfaAfG 3932 VPusUfsguaGfcGfUfa 2711 CCACCCTACAAGATACGC 3035 fAfuacgcuacsasa ucuUfgUfagggusgsg TACAT AD-1334486 ususccu(Ghd)GfuCfA 3933 VPusUfsgggUfcUfCfg 2712 TCTTCCTGGTCATCGAGA 3036 fUfcgagacccsasa augAfcCfaggaasgsa CCCAC AD-1334487 cscsagc(Ghd)UfgUfU 3934 VPusCfsaguCfgGfAfu 2713 GACCAGCGTGTTCATCCG 3037 fCfauccgacusgsa gaaCfaCfgcuggsusc ACTGC AD-1334488 csasgga(Chd)UfaCfA 3935 VPusAfscccUfgCfCfc 2714 ACCAGGACTACAAGGGCA 3038 fAfgggcagggsusa uugUfaGfuccugsgsu GGGTC AD-1334489 gsgsgaa(Chd)UfuCfG 3936 VPusGfscauUfgUfCfg 2715 GCGGGAACTTCGACGACA 3039 fAfcgacaaugscsa ucgAfaGfuucccsgsc ATGCC AD-1334490 csasaug(Ahd)CfuUfU 3937 VPusUfsacgCfgUfGfg 2716 ATCAATGACTTTGCCACG 3040 fGfccacgcgusasa caaAfgUfcauugsasu CGTAG AD-1334491 gscsacu(Ghd)GfaGfU 3938 VPusCfsuguUfcCfCfa 2717 ACGCACTGGAGTTTGGGA 3041 fUfugggaacasgsa aacUfcCfagugcsgsu ACAGC AD-1334492 gsgsccc(Ahd)GfaAfG 3939 VPusUfsgcuGfcAfCfu 2718 TGGGCCCAGAAGCAGTGC 3042 fCfagugcagcsasa gcuUfcUfgggccscsa AGCAT AD-1334493 cscsagg(Uhd)UfgAfC 3940 VPusAfscuuGfgUfGfg 2719 TCCCAGGTTGACTCCACC 3043 fUfccaccaagsusa aguCfaAfccuggsgsa AAGTA AD-1334494 csgsagg(Chd)CfuGfC 3941 VPusCfsgucGfuUfCfa 2720 TACGAGGCCTGCGTGAAC 3044 fGfugaacgacsgsa cgcAfgGfccucgsusa GACGC AD-1334495 ascsugc(Ghd)AfgUfG 3942 VPusCfsgugCfaGfAfa 2721 CGACTGCGAGTGTTTCTG 3045 fUfuucugcacsgsa acaCfuCfgcaguscsg CACGG AD-1334496 usgsugu(Ghd)UfgUfC 3943 VPusAfsgucCfgCfCfa 2722 CCTGTGTGTGTCCTGGCG 3046 fCfuggcggacsusa ggaCfaCfacacasgsg GACTC AD-1334497 usgsuuc(Uhd)GfuGfA 3944 VPusGfsuugUfaGfAfa 2723 CTTGTTCTGTGACTTCTA 3047 fCfuucuacaascsa gucAfcAfgaacasasg CAACC AD-1334498 usgsuga(Ghd)UfgGfC 3945 VPusGfsgcuGfgUfAfg 2724 GCTGTGAGTGGCACTACC 3048 fAfcuaccagcscsa ugcCfaCfucacasgsc AGCCC AD-1334499 gscsugc(Uhd)AfcCfC 3946 VPusUfsgggCfaCfUfu 2725 AGGCTGCTACCCGAAGTG 3049 fGfaagugcccsasa cggGfuAfgcagescsu CCCAC AD-1314302 asgsccc(Uhd)UfcUfU 3947 VPusGfsuccUfcAfUfu 2726 CCAGCCCTTCTTCAATGA 3050 fCfaaugaggascsa gaaGfaAfgggcusgsg GGACC AD-1334500 asasgug(Chd)GfuGfG 3948 VPusCfscacAfcUfGfg 2727 TGAAGTGCGTGGCCCAGT 3051 fCfccagugugsgsa gccAfcGfcacuuscsa GTGGC AD-1334501 csusacg(Ahd)CfaAfG 3949 VPusAfsguuUfcCfGfu 2728 TGCTACGACAAGGACGGA 3052 fGfacggaaacsusa ccuUfgUfcguagscsa AACTA AD-1334502 usgsacg(Uhd)CfgGfU 3950 VPusGfsgacCfcUfUfg 2729 TATGACGTCGGTGCAAGG 3053 fGfcaagggucscsa cacCfgAfcgucasusa GTCCC AD-1334503 cscsaga(Ghd)CfuGfU 3951 VPusGfsuguGfcAfGfu 2730 TGCCAGAGCTGTAACTGC 3054 fAfacugcacascsa uacAfgCfucuggscsa ACACC AD-1334504 csasgug(Chd)GfcUfC 3952 VPusUfscaaGfgCfUfg 2731 TCCAGTGCGCTCACAGCC 3055 fAfcagccuugsasa ugaGfcGfcacugsgsa TTGAG AD-1334505 csusgca(Chd)CfuAfU 3953 VPusUfsccuGfuCfCfu 2732 ACCTGCACCTATGAGGAC 3056 fGfaggacaggsasa cauAfgGfugcagsgsu AGGAC AD-1334506 csasgga(Chd)GfuCfA 3954 VPusGfsuguUfgUfAfg 2733 ACCAGGACGTCATCTACA 3057 fUfcuacaacascsa augAfcGfuccugsgsu ACACC AD-1334507 csgsccu(Ghd)CfuUfG 3955 VPusAfsgauGfgCfGfa 2734 GGCGCCTGCTTGATCGCC 3058 fAfucgccaucsusa ucaAfgCfaggcgscsc ATCTG AD-1334508 ascscau(Chd)AfuCfA 3956 VPusAfscagCfcUfUfc 2735 GCACCATCATCAGGAAGG 3059 fGfgaaggcugsusa cugAfuGfauggusgsc CTGTG AD-1334509 csascaa(Chd)GfcCfA 3957 VPusUfsgaaGfgUfGfa 2736 GCCACAACGCCATTCACC 3060 fUfucaccuucsasa augGfcGfuugugsgsc TTCAC AD-1334510 uscscac(Chd)GfuGfU 3958 VPusUfscgcGfgAfCfa 2737 TCTCCACCGTGTGTGTCC 3061 fGfuguccgcgsasa cacAfcGfguggasgsa GCGAG AD-1334511 uscscag(Chd)UfgGfU 3959 VPusUfsgccCfaUfUfg 2738 GGTCCAGCTGGTACAATG 3062 fAfcaaugggcsasa uacCfaGfcuggascsc GGCAC AD-1334512 csgsgag(Ahd)CfuUfU 3960 VPusCfsaaaCfgUfCfu 2739 GGCGGAGACTTTGAGACG 3063 fGfagacguuusgsa caaAfgUfcuccgscsc TTTGA AD-1334513 gsasggg(Uhd)AfcCfA 3961 VPusAfsgggCfaUfAfc 2740 GAGAGGGTACCAGGTATG 3064 fGfguaugcccsusa cugGfuAfcccucsusc CCCTG AD-1334514 csusggc(Uhd)GfaCfA 3962 VPusCfsggcAfcUfCfg 2741 TGCTGGCTGACATCGAGT 3065 fUfcgagugccsgsa augUfcAfgccagscsa GCCGG AD-1334515 csusucc(Chd)GfaCfA 3963 VPusUfsccaGfcGfGfc 2742 AGCTTCCCGACATGCCGC 3066 fUfgccgcuggsasa augUfcGfggaagscsu TGGAG AD-1334516 csasggu(Ghd)GfaCfU 3964 VPusAfsugcGfgUfCfa 2743 AGCAGGTGGACTGTGACC 3067 fGfugaccgcasusa cagUfcCfaccugscsu GCATG AD-1334517 csgscca(Ahd)CfaGfC 3965 VPusGfsacuCfuGfUfu 2744 TGCGCCAACAGCCAACAG 3068 fCfaacagaguscsa ggcUfgUfuggcgscsa AGTCC AD-1334518 uscsugu(Chd)AfcGfA 3966 VPusCfsagcUfcGfUfa 2745 GCTCTGTCACGACTACGA 3069 fCfuacgagcusgsa gucGfuGfacagasgsc GCTGC AD-1334519 uscsucu(Ghd)CfuGfC 3967 VPusGfscacGfuAfUfu 2746 GTTCTCTGCTGCGAATAC 3070 fGfaauacgugscsa cgcAfgCfagagasasc GTGCC AD-1334520 csascgg(Ahd)GfcCfU 3968 VPusUfsaggCfaCfAfg 2747 AGCACGGAGCCTGCTGTG 3071 fGfcugugccusasa cagGfcUfccgugscsu CCTAC AD-1334521 asgsacc(Ahd)CfaGfC 3969 VPusCfsuuuUfcGfGfu 2748 CCAGACCACAGCAACCGA 1445 fAfaccgaaaasgsa ugcUfgUfggucusgsg AAAGA AD-1334522 csasccu(Chd)GfcAfG 3970 VPusUfsggaCfcCfAfg 2749 CTCACCTCGCAGACTGGG 3072 fAfcuggguccsasa ucuGfcGfaggugsasg TCCAG AD-1334523 ascsaga(Ghd)UfgGfU 3971 VPusUfsccuCfaUfCfa 2750 GGACAGAGTGGTTTGATG 3073 fUfugaugaggsasa aacCfaCfucuguscsc AGGAC AD-1334524 gsascgu(Uhd)GfaGfU 3972 VPusUfsuauCfgUfAfg 2751 GGGACGTTGAGTCCTACG 3074 fCfcuacgauasasa gacUfcAfacgucscsc ATAAG AD-1334525 gsgsccg(Chd)UfgGfA 3973 VPusAfsuaaGfuGfCfc 2752 AGGGCCGCTGGAGGGCAC 3075 fGfggcacuuasusa cucCfaGfcggccscsu TTATG AD-1334526 csasgcc(Uhd)AfaGfG 3974 VPusCfsacuCfuAfUfg 2753 AGCAGCCTAAGGACATAG 3076 fAfcauagagusgsa uccUfuAfggcugscsu AGTGC AD-1334527 asascug(Ghd)AfcCfC 3975 VPusAfsccuGfuGfCfc 2754 CCAACTGGACCCTGGCAC 3077 fUfggcacaggsusa aggGfuCfcaguusgsg AGGTG AD-1334528 gsusgca(Chd)UfgUfG 3976 VPusAfsaguGfgAfCfg 2755 AGGTGCACTGTGACGTCC 3078 fAfcguccacususa ucaCfaGfugcacscsu ACTTC AD-1334529 gsusgca(Ghd)GfaAfC 3977 VPusCfscugCfuCfCfc 2756 GTGTGCAGGAACTGGGAG 3079 fUfgggagcagsgsa aguUfcCfugcacsasc CAGGA AD-1334530 csgsucu(Uhd)CfaAfG 3978 VPusUfsguaGfcAfCfa 2757 GGCGTCTTCAAGATGTGC 3080 fAfugugcuacsasa ucuUfgAfagacgscsc TACAA AD-1334531 csusgcu(Ghd)CfaGfU 3979 VPusAfsgugGfuCfGfu 2758 CTCTGCTGCAGTGACGAC 3081 fGfacgaccacsusa cacUfgCfagcagsasg CACTG AD-1334532 csgsacc(Ahd)CfaGfA 3980 VPusCfsgucUfcCfAfg 2759 ACCGACCACAGAGCTGGA 3082 fGfcuggagacsgsa cucUfgUfggucgsgsu GACGG AD-1334533 gscsccu(Ghd)UfuCfU 3981 VPusUfsgcgGfcGfUfu 2760 AGGCCCTGTTCTCAACGC 3083 fCfaacgccgcsasa gagAfaCfagggcscsu CGCAG AD-1334534 cscsucu(Chd)AfgAfA 3982 VPusAfsuguCfaGfUfc 2761 ACCCTCTCAGAAGGACTG 3084 fGfgacugacasusa cuuCfuGfagaggsgsu ACATC AD-1334535 csasgau(Ahd)CfaCfA 3983 VPusCfsaagGfgUfGfc 2762 CCCAGATACACAAGCACC 3085 fAfgcacccuusgsa uugUfgUfaucugsgsg CTTGG AD-1334536 gscsucc(Ahd)CfaGfA 3984 VPusGfsacaGfuGfGfg 2763 AGGCTCCACAGAACCCAC 3086 fAfcccacuguscsa uucUfgUfggagcscsu TGTCC AD-1334537 csasccc(Uhd)UfcCfA 3985 VPusCfsugaGfcGfUfg 2764 TCCACCCTTCCAACACGC 3087 fAfcacgcucasgsa uugGfaAfgggugsgsa TCAGC AD-1334538 csasaca(Ahd)CfaAfU 3986 VPusGfsgagGfuUfGfc 2765 CCCAACAACAATGGCAAC 3088 fGfgcaaccucscsa cauUfgUfuguugsgsg CTCCA AD-1334539 csgscuu(Chd)CfaAfA 3987 VPusUfscagCfgGfCfu 2766 ACCGCTTCCAAAGAGCCG 3089 fGfagccgcugsasa cuuUfgGfaagcgsgsu CTGAC AD-1334540 gscsgcc(Ahd)AfcAfC 3988 VPusUfscgcUfcGfUfg 2767 TGGCGCCAACACTCACGA 3090 fUfcacgagcgsasa aguGfuUfggcgcscsa GCGAG AD-1334541 gsuscca(Chd)CfuCfU 3989 VPusUfscucGfgCfCfu 2768 CTGTCCACCTCTCAGGCC 3091 fCfaggccgagsasa gagAfgGfuggacsasg GAGAC AD-1334542 csasgga(Chd)AfgAfG 3990 VPusUfscauUfgUfCfg 2769 CCCAGGACAGAGACGACA 3092 fAfcgacaaugsasa ucuCfuGfuccugsgsg ATGAG AD-1334543 csusuga(Chd)UfaAfC 3991 VPusUfsgguGfgUfGfg 2770 CCCTTGACTAACACCACC 3093 fAfccaccaccsasa uguUfaGfucaagsgsg ACCAG AD-1334544 csusguc(Ahd)AfcCfG 3992 VPusAfscucAfcAfCfu 2771 CGCTGTCAACCGAAGTGT 3094 fAfagugugagsusa ucgGfuUfgacagscsg GAGTG AD-1334545 asgsagu(Ghd)GfuUfU 3993 VPusAfsgucCfaCfGfu 2772 ACAGAGTGGTTTGACGTG 3095 fGfacguggacsusa caaAfcCfacucusgsu GACTT AD-1334546 gsgsaaa(Chd)UfuUfU 3994 VPusUfsgauGfuUfUfu 2773 ATGGAAACTTTTGAAAAC 3096 fGfaaaacaucsasa caaAfaGfuuuccsasu ATCAG AD-1334547 gscsacc(Ahd)AfaGfA 3995 VPusCfsacuCfuAfUfg 2774 GGGCACCAAAGAGCATAG 3097 fGfcauagagusgsa cucUfuUfggugcscsc AGTGC AD-1334548 csgsagg(Uhd)AfaGfC 3996 VPusCfscugGfuCfGfa 2775 CCCGAGGTAAGCATCGAC 3098 fAfucgaccagsgsa ugcUfuAfccucgsgsg CAGGT AD-1334549 csusgac(Chd)UfgCfA 3997 VPusGfsucuCfcAfGfg 2776 TGCTGACCTGCAGCCTGG 3099 fGfccuggagascsa cugCfaGfgucagscsa AGACG AD-1334550 csusgca(Ahd)GfaAfC 3998 VPusUfscugGfuCfUfu 2777 ACCTGCAAGAACGAAGAC 3100 fGfaagaccagsasa cguUfcUfugcagsgsu CAGAC AD-1334551 usgscuu(Chd)AfaCfU 3999 VPusCfsgcaCfgUfUfg 2778 TGTGCTTCAACTACAACG 3101 fAfcaacgugcsgsa uagUfuGfaagcascsa TGCGT AD-1334552 ususgcu(Ghd)UfgAfC 4000 VPusGfsgcuGfuAfGfu 2779 CTTTGCTGTGACGACTAC 3102 fGfacuacagcscsa cguCfaCfagcaasasg AGCCA AD-1334553 gsascga(Chd)CfuGfG 4001 VPusUfsuguGfaGfGfa 2780 GGGACGACCTGGATCCTC 3103 fAfuccucacasasa uccAfgGfucgucscsc ACAAA AD-1334554 csgsacc(Ahd)CfaAfC 4002 VPusCfsguaGfuGfGfc 2781 GCCGACCACAACAGCCAC 3104 fAfgccacuacsgsa uguUfgUfggucgsgsc TACGA AD-1334555 uscscac(Chd)CfuGfA 4003 VPusGfsgagCfuGfUfu 2782 CCTCCACCCTGAGAACAG 3105 fGfaacagcucscsa cucAfgGfguggasgsg CTCCC AD-1334556 uscscca(Ahd)AfgUfG 4004 VPusUfsgguGfgUfCfa 2783 CCTCCCAAAGTGCTGACC 3106 fCfugaccaccsasa gcaCfuUfugggasgsg ACCAC AD-1334557 csasgcu(Chd)CfaAfA 4005 VPusAfsgggAfgUfGfg 2784 ACCAGCTCCAAAGCCACT 3107 fGfccacucccsusa cuuUfgGfagcugsgsu CCCTC AD-1334558 cscsagu(Chd)CfaGfG 4006 VPusGfsguuGfcAfGfu 2785 CTCCAGTCCAGGGACTGC 3108 fGfacugcaacscsa cccUfgGfacuggsasg AACCG AD-1334559 asgscac(Uhd)GfaGfA 4007 VPusUfsggcUfgUfGfc 2786 CCAGCACTGAGAAGCACA 3109 fAfgcacagccsasa uucUfcAfgugcusgsg GCCAC AD-1334560 csusacc(Ahd)GfcGfU 4008 VPusGfsaugGfgUfGfu 2787 AGCTACCAGCGTTACACC 3110 fUfacacccauscsa aacGfcUfgguagscsu CATCC AD-1334561 ususccu(Chd)CfcUfG 4009 VPusAfsgguGfgUfGfc 2788 TCTTCCTCCCTGGGCACC 3111 fGfgcaccaccsusa ccaGfgGfaggaasgsa ACCTG AD-1334562 cscsuau(Chd)AfcAfG 4010 VPusGfsuguGfgUfGfg 2789 CGCCTATCACAGACCACC 3112 fAfccaccacascsa ucuGfuGfauaggscsg ACACC AD-1334563 cscsacc(Ahd)UfgUfC 4011 VPusUfsgugGfcUfGfu 2790 GGCCACCATGTCCACAGC 3113 fCfacagccacsasa ggaCfaUfgguggscsc CACAC AD-1334564 uscscuc(Chd)AfcUfC 4012 VPusGfscagUfcUfCfu 2791 CCTCCTCCACTCCAGAGA 3114 fCfagagacugscsa ggaGfuGfgaggasgsg CTGCC AD-1334565 csuscca(Chd)AfgUfG 4013 VPusUfsggcGfgUfAfa 2792 ACCTCCACAGTGCTTACC 3115 fCfuuaccgccsasa gcaCfuGfuggagsgsu GCCAC AD-1334566 csasgga(Ahd)CfaGfC 4014 VPusGfsguaGfuGfUfg 2793 CCCAGGAACAGCTCACAC 3116 fUfcacacuacscsa agcUfgUfuccugsgsg TACCA AD-1334567 usgscca(Ahd)CfuAfC 4015 VPusCfsgugGfuUfGfu 2794 AGTGCCAACTACCACAAC 3117 fCfacaaccacsgsa gguAfgUfuggcascsu CACGG AD-1334568 uscscag(Uhd)GfuGfG 4016 VPusUfsuguGfcUfGfa 2795 CCTCCAGTGTGGATCAGC 3118 fAfucagcacasasa uccAfcAfcuggasgsg ACAAC AD-1334569 ascscca(Chd)AfaCfC 4017 VPusUfsggaGfcCfUfc 2796 ACACCCACAACCAGAGGC 3119 fAfgaggcuccsasa uggUfuGfugggusgsu TCCAC AD-1334570 csgscca(Chd)AfgUfG 4018 VPusUfsgguGfgUfCfa 2797 ACCGCCACAGTGCTGACC 3120 fCfugaccaccsasa gcaCfuGfuggcgsgsu ACCAC AD-1334571 gscscac(Uhd)GfgUfU 4019 VPusGfsuugCfcAfUfa 2798 TGGCCACTGGTTCTATGG 3121 fCfuauggcaascsa gaaCfcAfguggcscsa CAACA AD-1334572 csusccu(Chd)UfaGfC 4020 VPusUfsgguCfuGfUfg 2799 CCCTCCTCTAGCACACAG 3122 fAfcacagaccsasa ugcUfaGfaggagsgsg ACCAG AD-1334573 gsgscca(Chd)UfaCfG 4021 VPusUfsggcCfgUfGfa 2800 ACGGCCACTACGATCACG 3123 fAfucacggccsasa ucgUfaGfuggccsgsu GCCAC AD-1334574 csusccu(Chd)AfaCfU 4022 VPusUfsuguCfcCfAfg 2801 CCCTCCTCAACTCCTGGG 3124 fCfcugggacasasa gagUfuGfaggagsgsg ACAAC AD-1334575 csasgca(Ahd)CfaCfA 4023 VPusAfsgggAfgUfCfa 2802 ACCAGCAACACAGTGACT 3125 fGfugacucccsusa cugUfgUfugcugsgsu CCCTC AD-1334576 usgsccc(Uhd)AfgGfG 4024 VPusUfsgugGfgUfGfg 2803 TCTGCCCTAGGGACCACC 3126 fAfccacccacsasa uccCfuAfgggcasgsa CACAC AD-1334577 asgsugc(Chd)GfaAfC 4025 VPusUfsggcCfaUfGfg 2804 CCAGTGCCGAACACCATG 3127 fAfccauggccsasa uguUfcGfgcacusgsg GCCAC AD-1334578 asgsccu(Ghd)GfaCfU 4026 VPusAfsgguGfgCfCfg 2805 ACAGCCTGGACTTCGGCC 3128 fUfcggccaccsusa aagUfcCfaggcusgsu ACCTC AD-1334579 ascscca(Chd)AfuCfA 4027 VPusGfsaagGfcUfCfu 2806 CCACCCACATCACAGAGC 3129 fCfagagccuuscsa gugAfuGfugggusgsg CTTCC AD-1334580 gsgsuga(Chd)UfuCfC 4028 VPusCfsuagGfgUfGfu 2807 ACGGTGACTTCCCACACC 3130 fCfacacccuasgsa gggAfaGfucaccsgsu CTAGC AD-1334581 csasacc(Ahd)CfcGfG 4029 VPusCfsuggGfuGfGfu 2808 AGCAACCACCGGTACCAC 3131 fUfaccacccasgsa accGfgUfgguugscsu CCAGC AD-1334582 csgsacu(Chd)CfaGfC 4030 VPusGfscugGfaAfAfg 2809 CTCGACTCCAGCCCTTTC 3132 fCfcuuuccagscsa ggcUfgGfagucgsasg CAGCC AD-1334583 usasgca(Ghd)CfaGfA 4031 VPusAfscucGfgUfGfg 2810 CCTAGCAGCAGAACCACC 3133 fAfccaccgagsusa uucUfgCfugcuasgsg GAGTC AD-1334584 ascscca(Ghd)CfaAfG 4032 VPusAfsgguGfcGfGfg 2811 ACACCCAGCAAGACCCGC 3134 fAfcccgcaccsusa ucuUfgCfugggusgsu ACCTC AD-1334585 csgsgug(Ghd)UfgAfC 4033 VPusAfscagCfcCfAfu 2812 CACGGTGGTGACCATGGG 3135 fCfaugggcugsusa gguCfaCfcaccgsusg CTGTG AD-1334586 gsusggc(Uhd)GfgAfC 4034 VPusGfsguaGfcUfGfu 2813 GAGTGGCTGGACTACAGC 3136 fUfacagcuacscsa aguCfcAfgccacsusc TACCC AD-1334587 ususgac(Ahd)CfcUfA 4035 VPusGfsaugUfuGfGfa 2814 CTTTGACACCTACTCCAA 3137 fCfuccaacauscsa guaGfgUfgucaasasg CATCC AD-1334588 ususggg(Chd)CfaGfG 4036 VPusCfsauuCfcAfCfg 2815 AGTTGGGCCAGGTCGTGG 3138 fUfcguggaausgsa accUfgGfcccaascsu AATGC AD-1334589 cscsugg(Ahd)CfuUfU 4037 VPusAfsgacCfaGfGfc 2816 AGCCTGGACTTTGGCCTG 3139 fGfgccuggucsusa caaAfgUfccaggscsu GTCTG AD-1334590 asusgug(Chd)UfuCfA 4038 VPusAfsuuuCfaUfAfg 2817 AGATGTGCTTCAACTATG 3140 fAfcuaugaaasusa uugAfaGfcacauscsu AAATC AD-1334591 usgsugu(Uhd)CfuGfC 4039 VPusCfsguaGfuUfGfc 2818 CGTGTGTTCTGCTGCAAC 3141 fUfgcaacuacsgsa agcAfgAfacacascsg TACGG AD-1334592 csasgcu(Chd)UfaCfG 4040 VPusAfsgggCfaUfGfg 2819 ACCAGCTCTACGGCCATG 3142 fGfccaugcccsusa ccgUfaGfagcugsgsu CCCTC AD-1334593 asusccu(Chd)AfcAfG 4041 VPusGfsuggUfcAfGfc 2820 GGATCCTCACAGAGCTGA 3143 fAfgcugaccascsa ucuGfuGfaggauscsc CCACA AD-1334594 cscsacu(Ahd)CfgAfC 4042 VPusAfsgugGfaCfUfc 2821 AGCCACTACGACTGAGTC 3144 fUfgaguccacsusa aguCfgUfaguggscsu CACTG AD-1334595 gsascca(Chd)CfuGfG 4043 VPusCfsuguGfaGfGfa 2822 GGGACCACCTGGATCCTC 3145 fAfuccucacasgsa uccAfgGfuggucscsc ACAGA AD-1334596 gsasgca(Chd)UfaCfA 4044 VPusUfscacGfgUfGfg 2823 CCGAGCACTACAGCCACC 3146 fGfccaccgugsasa cugUfaGfugcucsgsg GTGAC AD-1334597 cscsucc(Ahd)CfcCfA 4045 VPusAfsgcaGfuUfGfc 2824 CTCCTCCACCCAGGCAAC 3147 fGfgcaacugcsusa cugGfgUfggaggsasg TGCTG AD-1334598 gsgscca(Chd)GfaCfA 4046 VPusUfsgacUfgUfGfg 2825 ACGGCCACGACACCCACA 3148 fCfccacagucsasa gugUfcGfuggccsgsu GTCAC AD-1334599 gscsucc(Ahd)AfaGfC 4047 VPusGfsaagGfgAfGfu 2826 CAGCTCCAAAGCCACTCC 3149 fCfacucccuuscsa ggcUfuUfggagcsusg CTTCT AD-1334559 asgscac(Uhd)GfaGfA 4007 VPusUfsggcUfgUfGfc 2786 CCAGCACTGAGAAGCACA 3109 fAfgcacagccsasa uucUfcAfgugcusgsg GCCAC AD-1334600 ascsagc(Uhd)AfcCfA 4048 VPusGfscugUfaAfAfg 2827 CCACAGCTACCAGCTTTA 3150 fGfcuuuacagscsa cugGfuAfgcugusgsg CAGCC AD-1334562 cscsuau(Chd)AfcAfG 4010 VPusGfsuguGfgUfGfg 2789 CGCCTATCACAGACCACC 3112 fAfccaccacascsa ucuGfuGfauaggscsg ACACC AD-1334563 cscsacc(Ahd)UfgUfC 4011 VPusUfsgugGfcUfGfu 2790 GGCCACCATGTCCACAGC 3113 fCfacagccacsasa ggaCfaUfgguggscsc CACAC AD-1334601 uscscuc(Chd)AfcUfC 4049 VPusAfscagUfcUfCfu 2828 CCTCCTCCACTCCAGAGA 3151 fCfagagacugsusa ggaGfuGfgaggasgsg CTGTC AD-1334602 cscsaca(Ghd)UfgCfU 4050 VPusCfsgugGfuGfGfu 2829 CTCCACAGTGCTTACCAC 3152 fUfaccaccacsgsa aagCfaCfuguggsasg CACGG AD-1334603 gscsuca(Chd)AfcUfA 4051 VPusAfsgcaCfuUfUfg 2830 CAGCTCACACTACCAAAG 3153 fCfcaaagugcsusa guaGfuGfugagcsusg TGCTG AD-1334604 usascca(Chd)AfaCfC 4052 VPusUfsgaaGfcCfCfg 2831 ACTACCACAACCACGGGC 3154 fAfcgggcuucsasa uggUfuGfugguasgsu TTCAC AD-1334605 csascgc(Uhd)UfcCfA 4053 VPusUfsgauCfcAfCfa 2832 CGCACGCTTCCAGTGTGG 3155 fGfuguggaucsasa cugGfaAfgcgugscsg ATCAG AD-1334606 ascscca(Chd)AfaCfC 4054 VPusUfsggaAfcCfUfc 2833 ACACCCACAACCAGAGGT 3156 fAfgagguuccsasa uggUfuGfugggusgsu TCCAC AD-1334607 usgsacc(Ahd)CfcAfC 4055 VPusAfsguuGfuGfGfu 2834 GCTGACCACCACCACCAC 3157 fCfaccacaacsusa gguGfgUfggucasgsc AACTG AD-1334571 gscscac(Uhd)GfgUfU 4019 VPusGfsuugCfcAfUfa 2798 TGGCCACTGGTTCTATGG 3121 fCfuauggcaascsa gaaCfcAfguggcscsa CAACA AD-1334572 csusccu(Chd)UfaGfC 4020 VPusUfsgguCfuGfUfg 2799 CCCTCCTCTAGCACACAG 3122 fAfcacagaccsasa ugcUfaGfaggagsgsg ACCAG AD-1334573 gsgscca(Chd)UfaCfG 4021 VPusUfsggcCfgUfGfa 2800 ACGGCCACTACGATCACG 3123 fAfucacggccsasa ucgUfaGfuggccsgsu GCCAC AD-1334608 csusccu(Chd)AfaCfU 4056 VPusUfsuguCfcCfUfg 2835 CCCTCCTCAACTCCAGGG 3158 fCfcagggacasasa gagUfuGfaggagsgsg ACAAC AD-1334609 csasgca(Ghd)CfaCfA 4057 VPusAfsgggAfgUfCfa 2836 ACCAGCAGCACAGTGACT 3159 fGfugacucccsusa cugUfgCfugcugsgsu CCCTC AD-1334576 usgsccc(Uhd)AfgGfG 4024 VPusUfsgugGfgUfGfg 2803 TCTGCCCTAGGGACCACC 3126 fAfccacccacsasa uccCfuAfgggcasgsa CACAC AD-1334610 csascac(Ahd)CfgGfG 4058 VPusAfscagGfgAfUfc 2837 ACCACACACGGGCGATCC 3160 fCfgaucccugsusa gccCfgUfgugugsgsu CTGTC AD-1334578 asgsccu(Ghd)GfaCfU 4026 VPusAfsgguGfgCfCfg 2805 ACAGCCTGGACTTCGGCC 3128 fUfcggccaccsusa aagUfcCfaggcusgsu ACCTC AD-1334579 ascscca(Chd)AfuCfA 4027 VPusGfsaagGfcUfCfu 2806 CCACCCACATCACAGAGC 3129 fCfagagccuuscsa gugAfuGfugggusgsg CTTCC AD-1334611 cscsacc(Chd)AfgCfA 4059 VPusUfsggaGfuCfGfa 2838 TACCACCCAGCACTCGAC 3161 fCfucgacuccsasa gugCfuGfgguggsusa TCCAG AD-1334612 csasgcc(Chd)UfcAfC 4060 VPusUfsgcuGfcUfAfg 2839 TCCAGCCCTCACCCTAGC 3162 fCfcuagcagcsasa gguGfaGfggcugsgsa AGCAG AD-1334584 ascscca(Ghd)CfaAfG 4032 VPusAfsgguGfcGfGfg 2811 ACACCCAGCAAGACCCGC 3134 fAfcccgcaccsusa ucuUfgCfugggusgsu ACCTC AD-1334585 csgsgug(Ghd)UfgAfC 4033 VPusAfscagCfcCfAfu 2812 CACGGTGGTGACCATGGG 3135 fCfaugggcugsusa gguCfaCfcaccgsusg CTGTG AD-1334586 gsusggc(Uhd)GfgAfC 4034 VPusGfsguaGfcUfGfu 2813 GAGTGGCTGGACTACAGC 3136 fUfacagcuacscsa aguCfcAfgccacsusc TACCC AD-1334587 ususgac(Ahd)CfcUfA 4035 VPusGfsaugUfuGfGfa 2814 CTTTGACACCTACTCCAA 3137 fCfuccaacauscsa guaGfgUfgucaasasg CATCC AD-1334588 ususggg(Chd)CfaGfG 4036 VPusCfsauuCfcAfCfg 2815 AGTTGGGCCAGGTCGTGG 3138 fUfcguggaausgsa accUfgGfcccaascsu AATGC AD-1334589 cscsugg(Ahd)CfuUfU 4037 VPusAfsgacCfaGfGfc 2816 AGCCTGGACTTTGGCCTG 3139 fGfgccuggucsusa caaAfgUfccaggscsu GTCTG AD-1334590 asusgug(Chd)UfuCfA 4038 VPusAfsuuuCfaUfAfg 2817 AGATGTGCTTCAACTATG 3140 fAfcuaugaaasusa uugAfaGfcacauscsu AAATC AD-1334591 usgsugu(Uhd)CfuGfC 4039 VPusCfsguaGfuUfGfc 2818 CGTGTGTTCTGCTGCAAC 3141 fUfgcaacuacsgsa agcAfgAfacacascsg TACGG AD-1334613 csusgga(Uhd)CfcUfC 4061 VPusUfscugCfuCfUfg 2840 ACCTGGATCCTCACAGAG 3163 fAfcagagcagsasa ugaGfgAfuccagsgsu CAGAC AD-1334614 csasgcc(Ahd)CfuAfC 4062 VPusGfsguuGfcGfGfu 2841 AGCAGCCACTACGACCGC 3164 fGfaccgcaacscsa cguAfgUfggcugscsu AACCA AD-1334615 uscscca(Ahd)AfgUfG 4063 VPusUfsgcuGfgUfCfa 2842 CCTCCCAAAGTGCTGACC 3165 fCfugaccagcsasa gcaCfuUfugggasgsg AGCAC AD-1334616 csasguu(Chd)CfaAfA 4064 VPusAfsggaAfgUfGfg 2843 ACCAGTTCCAAAGCCACT 3166 fGfccacuuccsusa cuuUfgGfaacugsgsu TCCTC AD-1334617 csasagg(Ahd)CfuGfC 4065 VPusAfsaggGfuGfGfu 2844 TCCAAGGACTGCAACCAC 3167 fAfaccacccususa ugcAfgUfccuugsgsa CCTTC AD-1334618 gsusgcu(Ghd)AfcAfA 4066 VPusGfsuggCfuGfUfg 2845 CAGTGCTGACAAGCACAG 3168 fGfcacagccascsa cuuGfuCfagcacsusg CCACC AD-1334619 csascag(Chd)UfaCfC 4067 VPusGfsuguAfaAfGfc 2846 TCCACAGCTACCAGCTTT 3169 fAfgcuuuacascsa uggUfaGfcugugsgsa ACACC AD-1334620 csusccu(Uhd)CfaCfC 4068 VPusUfsgguCfcCfAfa 2847 CCCTCCTTCACCCTTGGG 3170 fCfuugggaccsasa gggUfgAfaggagsgsg ACCAC AD-1334621 cscscag(Ahd)AfcAfG 4069 VPusGfsuguGfgUfGfg 2848 CTCCCAGAACAGACCACC 3171 fAfccaccacascsa ucuGfuUfcugggsasg ACACC AD-1334622 csascca(Uhd)GfuCfC 4070 VPusGfsgugGfaUfUfg 2849 GCCACCATGTCCACAATC 3172 fAfcaauccacscsa uggAfcAfuggugsgsc CACCC AD-1334623 csuscca(Chd)AfgUfG 4071 VPusUfscguGfgUfCfa 2850 ACCTCCACAGTGCTGACC 3173 fCfugaccacgsasa gcaCfuGfuggagsgsu ACGAA AD-1334624 gsgscca(Chd)CfaGfU 4072 VPusUfsggaCfaUfGfg 2851 AGGGCCACCAGTTCCATG 3174 fUfccauguccsasa aacUfgGfuggccscsu TCCAC AD-1334593 asusccu(Chd)AfcAfG 4041 VPusGfsuggUfcAfGfc 2820 GGATCCTCACAGAGCTGA 3143 fAfgcugaccascsa ucuGfuGfaggauscsc CCACA AD-1334625 cscsacu(Ahd)CfaAfC 4073 VPusAfsgugGfcUfGfc 2852 AGCCACTACAACTGCAGC 3175 fUfgcagccacsusa aguUfgUfaguggscsu CACTG AD-1334595 gsascca(Chd)CfuGfG 4043 VPusCfsuguGfaGfGfa 2822 GGGACCACCTGGATCCTC 3145 fAfuccucacasgsa uccAfgGfuggucscsc ACAGA AD-1334626 csasgca(Chd)UfaCfA 4074 VPusUfscacGfgUfGfg 2853 CCCAGCACTACAGCCACC 3176 fGfccaccgugsasa cugUfaGfugcugsgsg GTGAC AD-1334627 ascsccu(Chd)AfaAfG 4075 VPusCfsuggUfcAfGfc 2854 GCACCCTCAAAGTGCTGA 3177 fUfgcugaccasgsa acuUfuGfagggusgsc CCAGC AD-1334628 ascscca(Chd)AfgUfC 4076 VPusUfsggaGfcUfGfa 2855 ACACCCACAGTCATCAGC 3178 fAfucagcuccsasa ugaCfuGfugggusgsu TCCAG AD-1334629 cscsacu(Chd)CfcUfC 4077 VPusUfsggaCfuGfGfa 2856 AGCCACTCCCTCCTCCAG 3179 fCfuccaguccsasa ggaGfgGfaguggscsu TCCAG AD-1334559 asgscac(Uhd)GfaGfA 4007 VPusUfsggcUfgUfGfc 2786 CCAGCACTGAGAAGCACA 3109 fAfgcacagccsasa uucUfcAfgugcusgsg GCCAC AD-1334630 csusacc(Ahd)GfcGfU 4078 VPusGfsaugGfcUfGfu 2857 AGCTACCAGCGTTACAGC 3180 fUfacagccauscsa aacGfcUfgguagscsu CATCC AD-1334562 cscsuau(Chd)AfcAfG 4010 VPusGfsuguGfgUfGfg 2789 CGCCTATCACAGACCACC 3112 fAfccaccacascsa ucuGfuGfauaggscsg ACACC AD-1334563 cscsacc(Ahd)UfgUfC 4011 VPusUfsgugGfcUfGfu 2790 GGCCACCATGTCCACAGC 3113 fCfacagccacsasa ggaCfaUfgguggscsc CACAC AD-1334631 cscsucu(Ahd)CfuCfC 4079 VPusGfsacaGfuCfUfc 2858 CTCCTCTACTCCAGAGAC 3181 fAfgagacuguscsa uggAfgUfagaggsasg TGTCC AD-1334602 cscsaca(Ghd)UfgCfU 4050 VPusCfsgugGfuGfGfu 2829 CTCCACAGTGCTTACCAC 3182 fUfaccaccacsgsa aagCfaCfuguggsasg CACGA AD-1334632 asgscuc(Ahd)CfaCfU 4080 VPusGfscacUfuUfGfg 2859 ACAGCTCACACTACCAAA 3183 fAfccaaagugscsa uagUfgUfgagcusgsu GTGCC AD-1334633 csusacc(Ahd)CfaAfC 4081 VPusGfsaagCfcCfGfu 2860 GACTACCACAACCACGGG 3184 fCfacgggcuuscsa gguUfgUfgguagsusc CTTCA AD-1334568 uscscag(Uhd)GfuGfG 4016 VPusUfsuguGfcUfGfa 2795 CCTCCAGTGTGGATCAGC 3118 fAfucagcacasasa uccAfcAfcuggasgsg ACAAC AD-1334569 ascscca(Chd)AfaCfC 4017 VPusUfsggaGfcCfUfc 2796 ACACCCACAACCAGAGGC 3119 fAfgaggcuccsasa uggUfuGfugggusgsu TCCAC AD-1334570 csgscca(Chd)AfgUfG 4018 VPusUfsgguGfgUfCfa 2797 ACCGCCACAGTGCTGACC 3120 fCfugaccaccsasa gcaCfuGfuggcgsgsu ACCAC AD-1334571 gscscac(Uhd)GfgUfU 4019 VPusGfsuugCfcAfUfa 2798 TGGCCACTGGTTCTATGG 3121 fCfuauggcaascsa gaaCfcAfguggcscsa CAACA AD-1334572 csusccu(Chd)UfaGfC 4020 VPusUfsgguCfuGfUfg 2799 CCCTCCTCTAGCACACAG 3122 fAfcacagaccsasa ugcUfaGfaggagsgsg ACCAG AD-1334634 gsgscca(Chd)UfaCfG 4082 VPusUfsggcUfgUfGfa 2861 ACGGCCACTACGATCACA 3185 fAfucacagccsasa ucgUfaGfuggccsgsu GCCAC AD-1334608 csusccu(Chd)AfaCfU 4056 VPusUfsuguCfcCfUfg 2835 CCCTCCTCAACTCCAGGG 3158 fCfcagggacasasa gagUfuGfaggagsgsg ACAAC AD-1334609 csasgca(Ghd)CfaCfA 4057 VPusAfsgggAfgUfCfa 2836 ACCAGCAGCACAGTGACT 3159 fGfugacucccsusa cugUfgCfugcugsgsu CCCTC AD-1334576 usgsccc(Uhd)AfgGfG 4024 VPusUfsgugGfgUfGfg 2803 TCTGCCCTAGGGACCACC 3126 fAfccacccacsasa uccCfuAfgggcasgsa CACAC AD-1334635 csascca(Chd)GfgCfC 4083 VPusCfsgugUfgUfGfg 2862 AACACCACGGCCACCACA 1551 fAfccacacacsgsa uggCfcGfuggugsusu CACGG AD-1334578 asgsccu(Ghd)GfaCfU 4026 VPusAfsgguGfgCfCfg 2805 ACAGCCTGGACTTCGGCC 3128 fUfcggccaccsusa aagUfcCfaggcusgsu ACCTC AD-1334579 ascscca(Chd)AfuCfA 4027 VPusGfsaagGfcUfCfu 2806 CCACCCACATCACAGAGC 3129 fCfagagccuuscsa gugAfuGfugggusgsg CTTCC AD-1334636 csasgca(Ghd)CfaAfC 4084 VPusGfsguaCfuGfGfu 2863 CCCAGCAGCAACCACCAG 3186 fCfaccaguacscsa gguUfgCfugcugsgsg TACCA AD-1334612 csasgcc(Chd)UfcAfC 4060 VPusUfsgcuGfcUfAfg 2839 TCCAGCCCTCACCCTAGC 3162 fCfcuagcagcsasa gguGfaGfggcugsgsa AGCAG AD-1334637 cscsucc(Ahd)GfgAfC 4085 VPusUfsgugGfcUfGfu 2864 CACCTCCAGGACCACAGC 3187 fCfacagccacsasa gguCfcUfggaggsusg CACAG AD-1334584 ascscca(Ghd)CfaAfG 4032 VPusAfsgguGfcGfGfg 2811 ACACCCAGCAAGACCCGC 3134 fAfcccgcaccsusa ucuUfgCfugggusgsu ACCTC AD-1334638 ascscac(Ghd)GfuGfG 4086 VPusCfsccgUfgGfUfc 2865 TAACCACGGTGGTGACCA 3188 fUfgaccacggsgsa accAfcCfguggususa CGGGC AD-1334586 gsusggc(Uhd)GfgAfC 4034 VPusGfsguaGfcUfGfu 2813 GAGTGGCTGGACTACAGC 3136 fUfacagcuacscsa aguCfcAfgccacsusc TACCC AD-1334587 ususgac(Ahd)CfcUfA 4035 VPusGfsaugUfuGfGfa 2814 CTTTGACACCTACTCCAA 3137 fCfuccaacauscsa guaGfgUfgucaasasg CATCC AD-1334588 ususggg(Chd)CfaGfG 4036 VPusCfsauuCfcAfCfg 2815 AGTTGGGCCAGGTCGTGG 3138 fUfcguggaausgsa accUfgGfcccaascsu AATGC AD-1334589 cscsugg(Ahd)CfuUfU 4037 VPusAfsgacCfaGfGfc 2816 AGCCTGGACTTTGGCCTG 3139 fGfgccuggucsusa caaAfgUfccaggscsu GTCTG AD-1334590 asusgug(Chd)UfuCfA 4038 VPusAfsuuuCfaUfAfg 2817 AGATGTGCTTCAACTATG 3140 fAfcuaugaaasusa uugAfaGfcacauscsu AAATC AD-1334591 usgsugu(Uhd)CfuGfC 4039 VPusCfsguaGfuUfGfc 2818 CGTGTGTTCTGCTGCAAC 3141 fUfgcaacuacsgsa agcAfgAfacacascsg TACGG AD-1334639 csgsgcc(Ahd)CfgCfC 4087 VPusAfsguuGfaGfGfa 2866 TACGGCCACGCCCTCCTC 3189 fCfuccucaacsusa gggCfgUfggccgsusa AACTC AD-1334640 csusgga(Uhd)CfcUfC 4088 VPusUfscagCfuUfUfg 2867 ACCTGGATCCTCACAAAG 3190 fAfcaaagcugsasa ugaGfgAfuccagsgsu CTGAC AD-1334594 cscsacu(Ahd)CfgAfC 4042 VPusAfsgugGfaCfUfc 2821 AGCCACTACGACTGAGTC 3144 fUfgaguccacsusa aguCfgUfaguggscsu CACTG AD-1334595 gsascca(Chd)CfuGfG 4043 VPusCfsuguGfaGfGfa 2822 GGGACCACCTGGATCCTC 3145 fAfuccucacasgsa uccAfgGfuggucscsc ACAGA AD-1334596 gsasgca(Chd)UfaCfA 4044 VPusUfscacGfgUfGfg 2823 CCGAGCACTACAGCCACC 3146 fGfccaccgugsasa cugUfaGfugcucsgsg GTGAC AD-1334597 cscsucc(Ahd)CfcCfA 4045 VPusAfsgcaGfuUfGfc 2824 CTCCTCCACCCAGGCAAC 3147 fGfgcaacugcsusa cugGfgUfggaggsasg TGCTG AD-1334598 gsgscca(Chd)GfaCfA 4046 VPusUfsgacUfgUfGfg 2825 ACGGCCACGACACCCACA 3148 fCfccacagucsasa gugUfcGfuggccsgsu GTCAC AD-1334599 gscsucc(Ahd)AfaGfC 4047 VPusGfsaagGfgAfGfu 2826 CAGCTCCAAAGCCACTCC 3149 fCfacucccuuscsa ggcUfuUfggagcsusg CTTCT AD-1334559 asgscac(Uhd)GfaGfA 4007 VPusUfsggcUfgUfGfc 2786 CCAGCACTGAGAAGCACA 3109 fAfgcacagccsasa uucUfcAfgugcusgsg GCCAC AD-1334600 ascsagc(Uhd)AfcCfA 4048 VPusGfscugUfaAfAfg 2827 CCACAGCTACCAGCTTTA 3150 fGfcuuuacagscsa cugGfuAfgcugusgsg CAGCC AD-1334562 cscsuau(Chd)AfcAfG 4010 VPusGfsuguGfgUfGfg 2789 CGCCTATCACAGACCACC 3112 fAfccaccacascsa ucuGfuGfauaggscsg ACACC AD-1334563 cscsacc(Ahd)UfgUfC 4011 VPusUfsgugGfcUfGfu 2790 GGCCACCATGTCCACAGC 3113 fCfacagccacsasa ggaCfaUfgguggscsc CACAC AD-1334564 uscscuc(Chd)AfcUfC 4012 VPusGfscagUfcUfCfu 2791 CCTCCTCCACTCCAGAGA 3114 fCfagagacugscsa ggaGfuGfgaggasgsg CTGCC AD-1334602 cscsaca(Ghd)UfgCfU 4050 VPusCfsgugGfuGfGfu 2829 CTCCACAGTGCTTACCAC 3152 fUfaccaccacsgsa aagCfaCfuguggsasg CACGG AD-1334632 asgscuc(Ahd)CfaCfU 4080 VPusGfscacUfuUfGfg 2859 ACAGCTCACACTACCAAA 3183 fAfccaaagugscsa uagUfgUfgagcusgsu GTGCC AD-1334633 csusacc(Ahd)CfaAfC 4081 VPusGfsaagCfcCfGfu 2860 GACTACCACAACCACGGG 3184 fCfacgggcuuscsa gguUfgUfgguagsusc CTTCA AD-1334568 uscscag(Uhd)GfuGfG 4016 VPusUfsuguGfcUfGfa 2795 CCTCCAGTGTGGATCAGC 3118 fAfucagcacasasa uccAfcAfcuggasgsg ACAAC AD-1334641 ascscca(Chd)AfaCfC 4089 VPusUfsggaGfcCfAfc 2868 ACACCCACAACCAGTGGC 3191 fAfguggcuccsasa uggUfuGfugggusgsu TCCAC AD-1334607 usgsacc(Ahd)CfcAfC 4055 VPusAfsguuGfuGfGfu 2834 GCTGACCACCACCACCAC 3157 fCfaccacaacsusa gguGfgUfggucasgsc AACTG AD-1334571 gscscac(Uhd)GfgUfU 4019 VPusGfsuugCfcAfUfa 2798 TGGCCACTGGTTCTATGG 3121 fCfuauggcaascsa gaaCfcAfguggcscsa CAACA AD-1334572 csusccu(Chd)UfaGfC 4020 VPusUfsgguCfuGfUfg 2799 CCCTCCTCTAGCACACAG 3122 fAfcacagaccsasa ugcUfaGfaggagsgsg ACCAG AD-1334573 gsgscca(Chd)UfaCfG 4021 VPusUfsggcCfgUfGfa 2800 ACGGCCACTACGATCACG 3123 fAfucacggccsasa ucgUfaGfuggccsgsu GCCAC AD-1334608 csusccu(Chd)AfaCfU 4056 VPusUfsuguCfcCfUfg 2835 CCCTCCTCAACTCCAGGG 3158 fCfcagggacasasa gagUfuGfaggagsgsg ACAAC AD-1334609 csasgca(Ghd)CfaCfA 4057 VPusAfsgggAfgUfCfa 2836 ACCAGCAGCACAGTGACT 3159 fGfugacucccsusa cugUfgCfugcugsgsu CCCTC AD-1334576 usgsccc(Uhd)AfgGfG 4024 VPusUfsgugGfgUfGfg 2803 TCTGCCCTAGGGACCACC 3126 fAfccacccacsasa uccCfuAfgggcasgsa CACAC AD-1334610 csascac(Ahd)CfgGfG 4058 VPusAfscagGfgAfUfc 2837 ACCACACACGGGCGATCC 3160 fCfgaucccugsusa gccCfgUfgugugsgsu CTGTC AD-1334578 asgsccu(Ghd)GfaCfU 4026 VPusAfsgguGfgCfCfg 2805 ACAGCCTGGACTTCGGCC 3128 fUfcggccaccsusa aagUfcCfaggcusgsu ACCTC AD-1334579 ascscca(Chd)AfuCfA 4027 VPusGfsaagGfcUfCfu 2806 CCACCCACATCACAGAGC 3129 fCfagagccuuscsa gugAfuGfugggusgsg CTTCC AD-1334611 cscsacc(Chd)AfgCfA 4059 VPusUfsggaGfuCfGfa 2838 TACCACCCAGCACTCGAC 3161 fCfucgacuccsasa gugCfuGfgguggsusa TCCAG AD-1334612 csasgcc(Chd)UfcAfC 4060 VPusUfsgcuGfcUfAfg 2839 TCCAGCCCTCACCCTAGC 3162 fCfcuagcagcsasa gguGfaGfggcugsgsa AGCAG AD-1334584 ascscca(Ghd)CfaAfG 4032 VPusAfsgguGfcGfGfg 2811 ACACCCAGCAAGACCCGC 3134 fAfcccgcaccsusa ucuUfgCfugggusgsu ACCTC AD-1334638 ascscac(Ghd)GfuGfG 4086 VPusCfsccgUfgGfUfc 2865 TAACCACGGTGGTGACCA 3188 fUfgaccacggsgsa accAfcCfguggususa CGGGC AD-1334586 gsusggc(Uhd)GfgAfC 4034 VPusGfsguaGfcUfGfu 2813 GAGTGGCTGGACTACAGC 3136 fUfacagcuacscsa aguCfcAfgccacsusc TACCC AD-1334587 ususgac(Ahd)CfcUfA 4035 VPusGfsaugUfuGfGfa 2814 CTTTGACACCTACTCCAA 3137 fCfuccaacauscsa guaGfgUfgucaasasg CATCC AD-1334588 ususggg(Chd)CfaGfG 4036 VPusCfsauuCfcAfCfg 2815 AGTTGGGCCAGGTCGTGG 3138 fUfcguggaausgsa accUfgGfcccaascsu AATGC AD-1334589 cscsugg(Ahd)CfuUfU 4037 VPusAfsgacCfaGfGfc 2816 AGCCTGGACTTTGGCCTG 3139 fGfgccuggucsusa caaAfgUfccaggscsu GTCTG AD-1334590 asusgug(Chd)UfuCfA 4038 VPusAfsuuuCfaUfAfg 2817 AGATGTGCTTCAACTATG 3140 fAfcuaugaaasusa uugAfaGfcacauscsu AAATC AD-1334591 usgsugu(Uhd)CfuGfC 4039 VPusCfsguaGfuUfGfc 2818 CGTGTGTTCTGCTGCAAC 3141 fUfgcaacuacsgsa agcAfgAfacacascsg TACGG AD-1334592 csasgcu(Chd)UfaCfG 4040 VPusAfsgggCfaUfGfg 2819 ACCAGCTCTACGGCCATG 3142 fGfccaugcccsusa ccgUfaGfagcugsgsu CCCTC AD-1334642 gsascga(Chd)CfuGfG 4090 VPusCfsuguGfaGfGfa 2869 GGGACGACCTGGATCCTC 3192 fAfuccucacasgsa uccAfgGfucgucscsc ACAGA AD-1334643 usgsacc(Ahd)CfaAfC 4091 VPusCfsguaGfuGfGfc 2870 GCTGACCACAACAGCCAC 3193 fAfgccacuacsgsa uguUfgUfggucasgsc TACGA AD-1334644 asuscca(Chd)UfgGfA 4092 VPusUfsggcCfgUfGfg 2871 GCATCCACTGGATCCACG 3194 fUfccacggccsasa aucCfaGfuggausgsc GCCAC AD-1334645 csusccc(Ahd)AfaGfU 4093 VPusGfscugGfuCfAfg 2872 CCCTCCCAAAGTGCTGAC 3195 fGfcugaccagscsa cacUfuUfgggagsgsg CAGCC AD-1334616 csasguu(Chd)CfaAfA 4064 VPusAfsggaAfgUfGfg 2843 ACCAGTTCCAAAGCCACT 3166 fGfccacuuccsusa cuuUfgGfaacugsgsu TCCTC AD-1334617 csasagg(Ahd)CfuGfC 4065 VPusAfsaggGfuGfGfu 2844 TCCAAGGACTGCAACCAC 3167 fAfaccacccususa ugcAfgUfccuugsgsa CCTTC AD-1334646 cscsacc(Ahd)AfaUfC 4094 VPusGfsguaGfcUfGfu 2873 AGCCACCAAATCCACAGC 3196 fCfacagcuacscsa ggaUfuUfgguggscsu TACCA AD-1334621 cscscag(Ahd)AfcAfG 4069 VPusGfsuguGfgUfGfg 2848 CTCCCAGAACAGACCACC 3171 fAfccaccacascsa ucuGfuUfcugggsasg ACACC AD-1334622 csascca(Uhd)GfuCfC 4070 VPusGfsgugGfaUfUfg 2849 GCCACCATGTCCACAATC 3172 fAfcaauccacscsa uggAfcAfuggugsgsc CACCC AD-1334623 csuscca(Chd)AfgUfG 4071 VPusUfscguGfgUfCfa 2850 ACCTCCACAGTGCTGACC 3173 fCfugaccacgsasa gcaCfuGfuggagsgsu ACGAA AD-1334647 gsgscca(Chd)CfaGfU 4095 VPusUfsggaCfgUfGfg 2874 AGGGCCACCAGTTCCACG 3197 fUfccacguccsasa aacUfgGfuggccscsu TCCAC AD-1334593 asusccu(Chd)AfcAfG 4041 VPusGfsuggUfcAfGfc 2820 GGATCCTCACAGAGCTGA 3143 fAfgcugaccascsa ucuGfuGfaggauscsc CCACA AD-1334625 cscsacu(Ahd)CfaAfC 4073 VPusAfsgugGfcUfGfc 2852 AGCCACTACAACTGCAGC 3175 fUfgcagccacsusa aguUfgUfaguggscsu CACTG AD-1334595 gsascca(Chd)CfuGfG 4043 VPusCfsuguGfaGfGfa 2822 GGGACCACCTGGATCCTC 3145 fAfuccucacasgsa uccAfgGfuggucscsc ACAGA AD-1334643 usgsacc(Ahd)CfaAfC 4091 VPusCfsguaGfuGfGfc 2870 GCTGACCACAACAGCCAC 3193 fAfgccacuacsgsa uguUfgUfggucasgsc TACGA AD-1334595 gsascca(Chd)CfuGfG 4043 VPusCfsuguGfaGfGfa 2822 GGGACCACCTGGATCCTC 3145 fAfuccucacasgsa uccAfgGfuggucscsc ACAGA AD-1334596 gsasgca(Chd)UfaCfA 4044 VPusUfscacGfgUfGfg 2823 CCGAGCACTACAGCCACC 3146 fGfccaccgugsasa cugUfaGfugcucsgsg GTGAC AD-1334597 cscsucc(Ahd)CfcCfA 4045 VPusAfsgcaGfuUfGfc 2824 CTCCTCCACCCAGGCAAC 3147 fGfgcaacugcsusa cugGfgUfggaggsasg TGCTG AD-1334648 usgsagc(Ahd)CfcAfC 4096 VPusUfsgucGfuGfGfc 2875 TGTGAGCACCACGGCCAC 3198 fGfgccacgacsasa cguGfgUfgcucascsa GACAC AD-1334557 csasgcu(Chd)CfaAfA 4005 VPusAfsgggAfgUfGfg 2784 ACCAGCTCCAAAGCCACT 3107 fGfccacucccsusa cuuUfgGfagcugsgsu CCCTC AD-1334649 cscsagg(Ghd)AfcUfG 4097 VPusAfsgggCfaGfUfu 2876 GTCCAGGGACTGCAACTG 3199 fCfaacugcccsusa gcaGfuCfccuggsasc CCCTT AD-1334559 asgscac(Uhd)GfaGfA 4007 VPusUfsggcUfgUfGfc 2786 CCAGCACTGAGAAGCACA 3109 fAfgcacagccsasa uucUfcAfgugcusgsg GCCAC AD-1334600 ascsagc(Uhd)AfcCfA 4048 VPusGfscugUfaAfAfg 2827 CCACAGCTACCAGCTTTA 3150 fGfcuuuacagscsa cugGfuAfgcugusgsg CAGCC AD-1334562 cscsuau(Chd)AfcAfG 4010 VPusGfsuguGfgUfGfg 2789 CGCCTATCACAGACCACC 3112 fAfccaccacascsa ucuGfuGfauaggscsg ACACC AD-1334563 cscsacc(Ahd)UfgUfC 4011 VPusUfsgugGfcUfGfu 2790 GGCCACCATGTCCACAGC 3113 fCfacagccacsasa ggaCfaUfgguggscsc CACAC AD-1334601 uscscuc(Chd)AfcUfC 4049 VPusAfscagUfcUfCfu 2828 CCTCCTCCACTCCAGAGA 3151 fCfagagacugsusa ggaGfuGfgaggasgsg CTGTC AD-1334565 csuscca(Chd)AfgUfG 4013 VPusUfsggcGfgUfAfa 2792 ACCTCCACAGTGCTTACC 3115 fCfuuaccgccsasa gcaCfuGfuggagsgsu GCCAC AD-1334632 asgscuc(Ahd)CfaCfU 4080 VPusGfscacUfuUfGfg 2859 ACAGCTCACACTACCAAA 3183 fAfccaaagugscsa uagUfgUfgagcusgsu GTGCC AD-1334633 csusacc(Ahd)CfaAfC 4081 VPusGfsaagCfcCfGfu 2860 GACTACCACAACCACGGG 3184 fCfacgggcuuscsa gguUfgUfgguagsusc CTTCA AD-1334568 uscscag(Uhd)GfuGfG 4016 VPusUfsuguGfcUfGfa 2795 CCTCCAGTGTGGATCAGC 3118 fAfucagcacasasa uccAfcAfcuggasgsg ACAAC AD-1334641 ascscca(Chd)AfaCfC 4089 VPusUfsggaGfcCfAfc 2868 ACACCCACAACCAGTGGC 3191 fAfguggcuccsasa uggUfuGfugggusgsu TCCAC AD-1334650 csgscca(Ghd)AfgUfG 4098 VPusUfsgguGfgUfCfa 2877 ACCGCCAGAGTGCTGACC 3200 fCfugaccaccsasa gcaCfuCfuggcgsgsu ACCAC AD-1334571 gscscac(Uhd)GfgUfU 4019 VPusGfsuugCfcAfUfa 2798 TGGCCACTGGTTCTATGG 3121 fCfuauggcaascsa gaaCfcAfguggcscsa CAACA AD-1334572 csusccu(Chd)UfaGfC 4020 VPusUfsgguCfuGfUfg 2799 CCCTCCTCTAGCACACAG 3122 fAfcacagaccsasa ugcUfaGfaggagsgsg ACCAG AD-1334573 gsgscca(Chd)UfaCfG 4021 VPusUfsggcCfgUfGfa 2800 ACGGCCACTACGATCACG 3123 fAfucacggccsasa ucgUfaGfuggccsgsu GCCAC AD-1334651 csasggg(Ahd)CfaAfC 4099 VPusGfsgugAfuGfGfg 2878 TCCAGGGACAACACCCAT 3201 fAfcccaucacscsa uguUfgUfcccugsgsa CACCC AD-1334652 csuscca(Ahd)AfgCfC 4100 VPusAfsggaGfgAfAfg 2879 AGCTCCAAAGCCACTTCC 3202 fAfcuuccuccsusa uggCfuUfuggagscsu TCCTC AD-1334617 csasagg(Ahd)CfuGfC 4065 VPusAfsaggGfuGfGfu 2844 TCCAAGGACTGCAACCAC 3167 fAfaccacccususa ugcAfgUfccuugsgsa CCTTC AD-1334653 usgscug(Ahd)CfaAfG 4101 VPusUfsgugGfcUfGfu 2880 AGTGCTGACAAGCACAGC 3203 fCfacagccacsasa gcuUfgUfcagcascsu CACAA AD-1334619 csascag(Chd)UfaCfC 4067 VPusGfsuguAfaAfGfc 2846 TCCACAGCTACCAGCTTT 3169 fAfgcuuuacascsa uggUfaGfcugugsgsa ACACC AD-1334654 csuscca(Chd)CfcUfG 4102 VPusAfscguGfgUfCfc 2881 TCCTCCACCCTGTGGACC 3204 fUfggaccacgsusa acaGfgGfuggagsgsa ACGTG AD-1334655 cscscag(Chd)AfcAfG 4103 VPusGfsuguGfgUfGfg 2882 GTCCCAGCACAGACCACC 3205 fAfccaccacascsa ucuGfuGfcugggsasc ACACC AD-1334656 gsuscca(Chd)CfaUfG 4104 VPusGfsgauUfgUfGfg 2883 ATGTCCACCATGTCCACA 3206 fUfccacaaucscsa acaUfgGfuggacsasu ATCCA AD-1334657 csusccu(Chd)UfaCfU 4105 VPusUfsgguCfuCfUfg 2884 ACCTCCTCTACTCCAGAG 3207 fCfcagagaccsasa gagUfaGfaggagsgsu ACCAC AD-1334658 csuscca(Chd)AfgUfG 4106 VPusUfsgguGfgUfCfa 2885 ACCTCCACAGTGCTGACC 3208 fCfugaccaccsasa gcaCfuGfuggagsgsu ACCAC AD-1334659 gsgscca(Chd)CfaAfU 4107 VPusUfsggcCfgUfGfg 2886 AGGGCCACCAATTCCACG 3209 fUfccacggccsasa aauUfgGfuggccscsu GCCAC AD-1334660 usgsacc(Ahd)CfaAfC 4108 VPusUfsguaGfuGfGfc 2887 GCTGACCACAACAGCCAC 3210 fAfgccacuacsasa uguUfgUfggucasgsc TACAA AD-1334661 usgsgau(Chd)CfaCfG 4109 VPusAfscagGfgUfGfg 2888 ACTGGATCCACGGCCACC 3211 fGfccacccugsusa ccgUfgGfauccasgsu CTGTC AD-1334595 gsascca(Chd)CfuGfG 4043 VPusCfsuguGfaGfGfa 2822 GGGACCACCTGGATCCTC 3145 fAfuccucacasgsa uccAfgGfuggucscsc ACAGA AD-1334662 gsasgca(Chd)UfaUfA 4110 VPusUfscacGfgUfGfg 2889 CCGAGCACTATAGCCACC 3212 fGfccaccgugsasa cuaUfaGfugcucsgsg GTGAT AD-1334663 cscsacu(Chd)UfgGfG 4111 VPusGfsugaGfcUfGfu 2890 CTCCACTCTGGGAACAGC 3213 fAfacagcucascsa uccCfaGfaguggsasg TCACA AD-1334664 csasugg(Chd)CfaCfU 4112 VPusCfsuguGfgGfCfa 2891 ACCATGGCCACTATGCCC 3214 fAfugcccacasgsa uagUfgGfccaugsgsu ACAGC AD-1334665 usgsccu(Chd)CfaCfG 4113 VPusAfsgcuGfgGfAfa 2892 ACTGCCTCCACGGTTCCC 3215 fGfuucccagcsusa ccgUfgGfaggcasgsu AGCTC AD-1334666 gscscaa(Chd)CfuUfC 4114 VPusUfsggaCfaCfGfc 2893 CTGCCAACCTTCAGCGTG 3216 fAfgcguguccsasa ugaAfgGfuuggcsasg TCCAC AD-1334667 uscscuc(Chd)UfcAfG 4115 VPusGfsuggUfgAfGfg 2894 TGTCCTCCTCAGTCCTCA 3217 fUfccucaccascsa acuGfaGfgaggascsa CCACC AD-1334668 uscscca(Chd)UfuCfU 4116 VPusCfsaggGfaGfUfa 2895 GCTCCCACTTCTCTACTC 3218 fCfuacucccusgsa gagAfaGfugggasgsc CCTGC AD-1334669 gscsauu(Uhd)GfgAfC 4117 VPusGfsagaAfaAfAfc 2896 GGGCATTTGGACAGTTTT 3219 fAfguuuuucuscsa uguCfcAfaaugescsc TCTCG AD-1334670 gsasagu(Chd)AfuCfU 4118 VPusGfsucuUfaUfUfg 2897 GGGAAGTCATCTACAATA 3220 fAfcaauaagascsa uagAfuGfacuucscsc AGACC AD-1334671 csusgcc(Ahd)UfuUfC 4119 VPusAfscacUfgCfGfu 2898 GGCTGCCATTTCTACGCA 3221 fUfacgcagugsusa agaAfaUfggcagscsc GTGTG AD-1334672 csascug(Uhd)GfaCfA 4120 VPusAfsagcGfgUfCfa 2899 AGCACTGTGACATTGACC 3222 fUfugaccgcususa augUfcAfcagugscsu GCTTC AD-1334673 usgsuga(Chd)AfaUfG 4121 VPusAfsgagGfgAfUfg 2900 GCTGTGACAATGCCATCC 3223 fCfcaucccucsusa gcaUfuGfucacasgsc CTCTC AD-1334674 ascsccu(Ghd)GfaGfA 4122 VPusAfsccgUfgCfAfg 2901 GGACCCTGGAGAACTGCA 3224 fAfcugcacggsusa uucUfcCfaggguscsc CGGTG AD-1334675 gsusggg(Uhd)GfaCfA 4123 VPusAfscgaCfaCfGfg 2902 GCGTGGGTGACAACCGTG 3225 fAfccgugucgsusa uugUfcAfcccacsgsc TCGTC AD-1334676 gsasccc(Ahd)AfaGfC 4124 VPusUfsuggCfcAfCfa 2903 TGGACCCAAAGCCTGTGG 3226 fCfuguggccasasa ggcUfuUfgggucscsa CCAAC AD-1334677 csusgcg(Uhd)GfaAfC 4125 VPusGfscagGfuGfCfu 2904 ACCTGCGTGAACAAGCAC 3227 fAfagcaccugscsa uguUfcAfcgcagsgsu CTGCC AD-1334678 uscsaaa(Ghd)UfgUfC 4126 VPusGfscucGfgGfUfc 2905 CATCAAAGTGTCGGACCC 3228 fGfgacccgagscsa cgaCfaCfuuugasusg GAGCC AD-1334679 cscsugu(Ghd)AfcUfU 4127 VPusCfsucaUfaGfUfg 2906 GCCCTGTGACTTCCACTA 3229 fCfcacuaugasgsa gaaGfuCfacaggsgsc TGAGT AD-1334680 gsasgug(Chd)AfuCfU 4128 VPusCfsacaUfgCfUfg 2907 GCGAGTGCATCTGCAGCA 3230 fGfcagcaugusgsa cagAfuGfcacucsgsc TGTGG AD-1334681 cscscac(Uhd)AfuUfC 4129 VPusGfsucaAfaGfGfu 2908 CTCCCACTATTCCACCTT 3231 fCfaccuuugascsa ggaAfuAfgugggsasg TGACG AD-1334682 ascscua(Uhd)GfuCfC 4130 VPusUfscucUfcAfUfg 2909 GCACCTATGTCCTCATGA 3232 fUfcaugagagsasa aggAfcAfuaggusgsc GAGAG AD-1334683 csascgc(Uhd)UfuGfG 4131 VPusGfscugAfgAfUfu 2910 TGCACGCTTTGGGAATCT 3233 fGfaaucucagscsa cccAfaAfgcgugscsa CAGCC AD-1334684 csusgga(Chd)AfaCfC 4132 VPusGfsugcAfgUfAfg 2911 ACCTGGACAACCACTACT 3234 fAfcuacugcascsa uggUfuGfuccagsgsu GCACG AD-1334685 csuscag(Chd)AfuCfC 4133 VPusGfsacuUfgUfAfg 2912 CCCTCAGCATCCACTACA 3235 fAfcuacaaguscsa uggAfuGfcugagsgsg AGTCC AD-1334686 gsusccu(Chd)AfcUfG 4134 VPusAfsccaUfgGfUfg 2913 TCGTCCTCACTGTCACCA 3236 fUfcaccauggsusa acaGfuGfaggacsgsa TGGTG AD-1334687 cscsuga(Uhd)CfcUfG 4135 VPusUfsuugGfuCfAfa 2914 GGCCTGATCCTGTTTGAC 3237 fUfuugaccaasasa acaGfgAfucaggscsc CAAAT AD-1334688 asgscgg(Uhd)UfuCfA 4136 VPusCfscguUfcUfUfg 2915 GCAGCGGTTTCAGCAAGA 3238 fGfcaagaacgsgsa cugAfaAfccgcusgsc ACGGC AD-1334689 csgsugu(Ghd)GfaCfA 4137 VPusAfsgggCfaGfGfa 2916 TGCGTGTGGACATTCCTG 3239 fUfuccugcccsusa augUfcCfacacgscsa CCCTG AD-1334690 gsusgag(Chd)GfuCfA 4138 VPusCfscauUfgAfAfg 2917 GCGTGAGCGTCACCTTCA 3240 fCfcuucaaugsgsa gugAfcGfcucacsgsc ATGGC AD-1334691 asgsccu(Chd)UfuCfC 4139 VPusGfsuguUfgUfUfg 2918 ACAGCCTCTTCCACAACA 3241 fAfcaacaacascsa uggAfaGfaggcusgsu ACACC AD-1334692 usgscac(Chd)AfaCfA 4140 VPusUfscccUfcUfGfg 2919 CCTGCACCAACAACCAGA 3242 fAfccagagggsasa uugUfuGfgugcasgsg GGGAC AD-1334693 usgsucu(Chd)CfaGfC 4141 VPusGfsuucCfgUfCfc 2920 ACTGTCTCCAGCGGGACG 3243 fGfggacggaascsa cgcUfgGfagacasgsu GAACC AD-1334694 csgscca(Ghd)UfuGfC 4142 VPusCfscauGfuCfCfu 2921 GCCGCCAGTTGCAAGGAC 3244 fAfaggacaugsgsa ugcAfaCfuggcgsgsc ATGGC AD-1334695 csgsaca(Ghd)CfaGfA 4143 VPusAfsgccAfuCfCfu 2922 CCCGACAGCAGAAAGGAT 3245 fAfaggauggcsusa uucUfgCfugucgsgsg GGCTG AD-1334696 cscsgcu(Chd)UfgUfG 4144 VPusAfsgcaUfcAfGfa 2923 AGCCGCTCTGTGATCTGA 3246 fAfucugaugcsusa ucaCfaGfageggscsu TGCTG AD-1334697 csasggu(Chd)UfuUfG 4145 VPusUfsggcAfcUfCfa 2924 GCCAGGTCTTTGCTGAGT 3247 fCfugagugccsasa gcaAfaGfaccugsgsc GCCAC AD-1334698 gsgsgcc(Chd)AfuUfC 4146 VPusAfsggcGfuUfGfa 2925 CCGGGCCCATTCTTCAAC 3248 fUfucaacgccsusa agaAfuGfggcccsgsg GCCTG AD-1334699 gsasggc(Uhd)UfaCfG 4147 VPusCfsagaGfcUfCfu 2926 TGGAGGCTTACGCAGAGC 3249 fCfagagcucusgsa gcgUfaAfgccucscsa TCTGC AD-1334700 asgsugu(Ghd)CfaGfU 4148 VPusCfsucgCfcAfGfu 2927 GGAGTGTGCAGTGACTGG 3250 fGfacuggcgasgsa cacUfgCfacacuscsc CGAGG AD-1334701 cscsacc(Ahd)AfaGfU 4149 VPusUfsggcUfuGfUfa 2928 ACCCACCAAAGTGTACAA 3251 fGfuacaagccsasa cacUfuUfgguggsgsu GCCAT AD-1334702 csusgca(Ahd)CfuCfU 4150 VPusUfscugGfuUfCfc 2929 ACCTGCAACTCTAGGAAC 3252 fAfggaaccagsasa uagAfgUfugcagsgsu CAGAG AD-1334703 csasgau(Chd)CfuCfU 4151 VPusUfsgugCfgUfUfg 2930 ACCAGATCCTCTTCAACG 3253 fUfcaacgcacsasa aagAfgGfaucugsgsu CACAC AD-1334704 gsgsgca(Uhd)CfuGfC 4152 VPusAfsggcCfuGfCfa 2931 ATGGGCATCTGCGTGCAG 3254 fGfugcaggccsusa cgcAfgAfugcccsasu GCCTG AD-1334705 csgsaug(Ghd)GfuUfU 4153 VPusGfsaaaUfuUfAfg 2932 CCCGATGGGTTTCCTAAA 3255 fCfcuaaauuuscsa gaaAfcCfcaucgsgsg TTTCC AD-1334706 gsgsuca(Ghd)CfaAfC 4154 VPusAfsggaCfuGfGfc 2933 TGGGTCAGCAACTGCCAG 3256 fUfgccaguccsusa aguUfgCfugaccscsa TCCTG AD-1334707 gsasggg(Uhd)UfcAfG 4155 VPusUfsgcaCfcGfAfc 2934 ACGAGGGTTCAGTGTCGG 3257 fUfgucggugcsasa acuGfaAfcccucsgsu TGCAG AD-1334708 csgsgcu(Uhd)CfgUfA 4156 VPusUfsgguCfaCfGfg 2935 CCCGGCTTCGTAACCGTG 3258 fAfccgugaccsasa uuaCfgAfagccgsgsg ACCAG AD-1334709 csgsugu(Ghd)CfaAfC 4157 VPusAfsgguGfgUfUfg 2936 TGCGTGTGCAACACAACC 3259 fAfcaaccaccsusa uguUfgCfacacgscsa ACCTG AD-1334710 csasgga(Ghd)UfcCfA 4158 VPusUfsgggUfgCfAfg 2937 GGCAGGAGTCCATCTGCA 3260 fUfcugcacccsasa augGfaCfuccugscsc CCCAG AD-1334711 csusguc(Chd)CfaCfC 4159 VPusUfsgcaGfcGfGfa 2938 TGCTGTCCCACCTTCCGC 3261 fUfuccgcugcsasa aggUfgGfgacagscsa TGCAG AD-1334712 uscsagc(Uhd)GfuGfU 4160 VPusCfsauuGfuAfCfg 2939 CCTCAGCTGTGTTCGTAC 3262 fUfcguacaausgsa aacAfcAfgcugasgsg AATGG AD-1334713 ususggu(Ghd)CfaAfC 4161 VPusGfsccuGfgGfAfa 2940 GGTTGGTGCAACCTTCCC 3263 fCfuucccaggscsa gguUfgCfaccaascsc AGGCG AD-1334714 uscsccu(Ghd)CfcAfC 4162 VPusAfsgguAfcAfCfa 2941 CTTCCCTGCCACATGTGT 3264 fAfuguguaccsusa uguGfgCfagggasasg ACCTG AD-1334715 ascsggu(Ghd)CfaAfU 4163 VPusUfsccuCfcUfGfa 2942 CAACGGTGCAATGTCAGG 3265 fGfucaggaggsasa cauUfgCfaccgususg AGGAT AD-1334716 csusgca(Ahd)CfaAfU 4164 VPusGfsacaGfgUfAfg 2943 GCCTGCAACAATACTACC 3266 fAfcuaccuguscsa uauUfgUfugcagsgsc TGTCC AD-1334717 gsgsgcu(Uhd)UfgAfG 4165 VPusCfsucuCfuUfGfu 2944 CAGGGCTTTGAGTACAAG 3267 fUfacaagagasgsa acuCfaAfagcccsusg AGAGT AD-1334718 gsuscca(Ghd)CfuGfA 4166 VPusCfsaggUfuUfCfa 2945 CAGTCCAGCTGAATGAAA 3268 fAfugaaaccusgsa uucAfgCfuggacsusg CCTGG AD-1334719 csasaca(Ghd)CfcAfU 4167 VPusAfsguuGfuCfCfa 2946 GTCAACAGCCATGTGGAC 3269 fGfuggacaacsusa cauGfgCfuguugsasc AACTG AD-1334720 cscsgug(Uhd)AfcCfU 4168 VPusAfsgccUfcAfCfa 2947 CACCGTGTACCTCTGTGA 3270 fCfugugaggcsusa gagGfuAfcacggsusg GGCTG AD-1334721 gsgsugg(Ahd)GfuCfC 4169 VPusGfsucaGfcAfAfa 2948 AGGGTGGAGTCCATTTGC 3271 fAfuuugcugascsa uggAfcUfccaccscsu TGACC AD-1334722 csusgcc(Chd)AfgAfU 4170 VPusAfsgcuGfgAfCfa 2949 TCCTGCCCAGATGTGTCC 3272 fGfuguccagcsusa cauCfuGfggcagsgsa AGCTG AD-1334723 gscsugc(Uhd)AfcUfC 4171 VPusCfsuccUfcAfCfa 2950 CTGCTGCTACTCCTGTGA 3273 fCfugugaggasgsa ggaGfuAfgcagcsasg GGAGG AD-1334724 uscscug(Uhd)CfaAfG 4172 VPusUfsugaUfgCfGfg 2951 ACTCCTGTCAAGTCCGCA 3274 fUfccgcaucasasa acuUfgAfcaggasgsu TCAAC AD-1334725 gsascca(Uhd)CfcUfG 4173 VPusCfscugGfuGfCfc 2952 ACGACCATCCTGTGGCAC 3275 fUfggcaccagsgsa acaGfgAfuggucsgsu CAGGG AD-1320631 gsgsuca(Ahd)CfaUfC 4174 VPusCfsgcaGfaAfGfg 2953 GAGGTCAACATCACCTTC 3276 fAfccuucugcsgsa ugaUfgUfugaccsusc TGCGA AD-1334726 csgsucc(Ahd)AfgUfA 4175 VPusCfsucuGfcUfGfa 2954 AGCGTCCAAGTACTCAGC 3277 fCfucagcagasgsa guaCfuUfggacgscsu AGAGG AD-1334727 asusgca(Ghd)CfaCfC 4176 VPusCfsaggUfgCfAfc 2955 CCATGCAGCACCAGTGCA 3278 fAfgugcaccusgsa uggUfgCfugcausgsg CCTGC AD-1334728 gscsccu(Uhd)GfcAfC 4177 VPusCfsguuAfgGfAfc 2956 GTGCCCTTGCACTGTCCT 3279 fUfguccuaacsgsa aguGfcAfagggcsasc AACGG AD-1334729 csusgca(Chd)AfcCfU 4178 VPusAfscguGfgGfUfg 2957 TCCTGCACACCTACACCC 3280 fAfcacccacgsusa uagGfuGfugcagsgsa ACGTG AD-1334730 gscsacg(Chd)CfcUfU 4179 VPusAfsgggAfcAfCfa 2958 CTGCACGCCCTTCTGTGT 3281 fCfugugucccsusa gaaGfgGfcgugcsasg CCCTG AD-1334731 ascsugc(Uhd)GfuCfU 4180 VPusGfsaacGfuUfCfu 2959 CCACTGCTGTCTGAGAAC 3282 fGfagaacguuscsa cagAfcAfgcagusgsg GTTCT AD-1334732 csasugc(Uhd)CfuGfU 4181 VPusCfsuccAfgGfUfg 2960 CCCATGCTCTGTCCACCT 3283 fCfcaccuggasgsa gacAfgAfgcaugsgsg GGAGC AD-1334733 gscsauu(Ghd)UfcUfG 4182 VPusUfsuuuCfaUfGfa 2961 GTGCATTGTCTGATCATG 3284 fAfucaugaaasasa ucaGfaCfaaugcsasc AAAAC AD-1334734 gsgscgc(Chd)AfcUfC 4183 VPusUfsaggAfcUfCfc 2962 AGGGCGCCACTCAGGAGT 3285 fAfggaguccusasa ugaGfuGfgcgccscsu CCTAC AD-1334735 csusccc(Uhd)GfaUfG 4184 VPusGfsuccCfaGfUfg 2963 CCCTCCCTGATGTCACTG 3286 fUfcacugggascsa acaUfcAfgggagsgsg GGACG AD-1334736 csusgga(Ahd)CfaAfA 4185 VPusAfscauGfcUfUfa 2964 CCCTGGAACAAACTAAGC 3287 fCfuaagcaugsusa guuUfgUfuccagsgsg ATGTG AD-1334737 gscsacg(Ghd)AfuUfC 4186 VPusUfsggcCfaGfCfu 2965 CAGCACGGATTCCAGCTG 3288 fCfagcuggccsasa ggaAfuCfcgugcsusg GCCAC AD-1334738 gsascag(Ghd)CfuGfG 4187 VPusCfsuugCfcUfGfg 2966 CAGACAGGCTGGTCCAGG 3289 fUfccaggcaasgsa accAfgCfcugucsusg CAAGG AD-1334739 gscsugc(Chd)AfgGfA 4188 VPusUfsgucGfcAfGfc 2967 CTGCTGCCAGGAAGCTGC 3290 fAfgcugcgacsasa uucCfuGfgcagcsasg GACAG AD-1334740 gscsagg(Ghd)UfaAfC 4189 VPusUfscagCfcCfUfg 2968 CTGCAGGGTAACTCAGGG 3291 fUfcagggcugsasa aguUfaCfccugcsasg CTGAG AD-1334741 gscsaac(Ghd)GfcCfA 4190 VPusCfsucuCfuGfAfc 2969 TCGCAACGGCCAGGTCAG 3292 fGfgucagagasgsa cugGfcCfguugcsgsa AGAGG AD-1334742 asgsccc(Ahd)GfuUfU 4191 VPusUfsuuaUfuUfGfc 2970 CCAGCCCAGTTTTGCAAA 3293 fUfgcaaauaasasa aaaAfcUfgggcusgsg TAAAC

TABLE 8 MUC5B Single Dose In Vitro Screen in A549 Cells. MUC5B/gapdh 10 nM DUPLEX ID mean SD AD-1334420.1 0.273 0.030 AD-1334419.1 0.738 0.047 AD-1334418.1 0.408 0.050 AD-1334417.1 0.645 0.050 AD-1334416.1 0.653 0.078 AD-1334415.1 0.483 0.038 AD-1334414.1 0.260 0.028 AD-1334413.1 0.378 0.115 AD-1334412.1 0.604 0.108 AD-1334411.1 0.203 0.019 AD-1334410.1 0.377 0.057 AD-1334409.1 0.199 0.019 AD-1334408.1 0.247 0.044 AD-1334407.1 0.704 0.074 AD-1334406.1 0.657 0.085 AD-1334405.1 0.326 0.034 AD-1334404.1 0.240 0.012 AD-1334403.1 0.271 0.014 AD-1334402.1 0.670 0.032 AD-1334401.1 0.275 0.029 AD-1334400.1 0.274 0.092 AD-1334399.1 0.529 0.099 AD-1334398.1 0.370 0.073 AD-1334397.1 0.433 0.084 AD-1334396.1 0.356 0.042 AD-1334395.1 0.246 0.009 AD-1334394.1 0.245 0.037 AD-1334393.1 0.175 0.024 AD-1334392.1 0.382 0.063 AD-1334391.1 0.474 0.053 AD-1334390.1 0.335 0.011 AD-1334389.1 0.242 0.038 AD-1334388.1 0.386 0.033 AD-1334387.1 0.320 0.052 AD-1334386.1 0.302 0.022 AD-1334385.1 0.344 0.043 AD-1334384.1 0.241 0.041 AD-1334383.1 0.359 0.096 AD-1334382.1 0.179 0.080 AD-1334381.1 0.401 0.049 AD-1334380.1 0.351 0.006 AD-1334379.1 0.266 0.037 AD-1334378.1 0.435 0.073 AD-1334377.1 0.519 0.112 AD-1334376.1 0.330 0.042 AD-1334375.1 0.420 0.028 AD-1334374.1 0.270 0.032 AD-1334373.1 0.249 0.026 AD-1334372.1 0.544 0.035 AD-1334371.1 0.427 0.057 AD-1334370.1 0.702 0.060 AD-1334369.1 0.775 0.090 AD-1334368.1 0.234 0.032 AD-1334367.1 0.303 0.037 AD-1334366.1 0.386 0.054 AD-1334365.1 0.433 0.019 AD-1334364.1 0.199 0.062 AD-1334363.1 0.749 0.119 AD-1334362.1 0.474 0.089 AD-1334361.1 0.449 0.060 AD-1334360.1 0.359 0.043 AD-1334359.1 0.192 0.085 AD-1334358.1 0.322 0.017 AD-1334357.1 0.318 0.033 AD-1334356.1 0.269 0.010 AD-1334355.1 0.690 0.184 AD-1334354.1 0.266 0.055 AD-1334353.1 0.201 0.053 AD-1334352.1 0.343 0.037 AD-1334351.1 0.208 0.034 AD-1334350.1 0.290 0.025 AD-1334349.1 0.287 0.029 AD-1334348.1 0.293 0.118 AD-1334347.1 0.167 0.044 AD-1334346.1 0.206 0.071 AD-1334345.1 0.357 0.145 AD-1334344.1 0.566 0.199 AD-1334343.1 0.226 0.083 AD-1334342.1 0.377 0.181 AD-1334341.1 0.389 0.051 AD-1334340.1 0.655 0.029 AD-1334339.1 0.390 0.036 AD-1334272.1 0.404 0.038 AD-1334338.1 0.346 0.048 AD-1334337.1 0.467 0.073 AD-1334336.1 0.633 0.036 AD-1334335.1 0.350 0.033 AD-1334334.1 0.239 0.070 AD-1334333.1 0.433 0.133 AD-1334331.1 0.736 0.091 AD-1334296.1 0.243 0.085 AD-1334330.1 0.438 0.042 AD-1334294.1 0.346 0.015 AD-1334329.1 0.307 0.042 AD-1334328.1 0.632 0.160 AD-1334250.1 0.480 0.105 AD-1334249.1 0.324 0.036 AD-1334248.1 0.281 0.033 AD-1334327.1 0.428 0.047 AD-1334318.1 0.436 0.143 AD-1334245.1 0.336 0.058 AD-1334310.1 0.641 0.061 AD-1334309.1 0.371 0.112 AD-1334242.1 0.402 0.047 AD-1334278.1 0.383 0.027 AD-1334240.1 0.374 0.122 AD-1334277.1 0.299 0.095 AD-1334236.1 0.468 0.023 AD-1334326.1 0.662 0.062 AD-1334234.1 0.328 0.021 AD-1334325.1 0.739 0.165 AD-1334274.1 0.576 0.229 AD-1334273.1 0.412 0.021 AD-1334320.1 0.499 0.133 AD-1334302.1 0.630 0.018 AD-1334270.1 0.391 0.028 AD-1334324.1 0.293 0.052 AD-1334300.1 0.261 0.068 AD-1334299.1 0.284 0.007 AD-1334323.1 0.474 0.075 AD-1334293.1 0.239 0.026 AD-1334322.1 0.363 0.044 AD-1334321.1 0.606 0.040 AD-1334319.1 0.366 0.027 AD-1334269.1 0.523 0.030 AD-1334268.1 0.452 0.046 AD-1334267.1 0.232 0.033 AD-1334266.1 0.319 0.027 AD-1334265.1 0.563 0.005 AD-1334264.1 0.368 0.033 AD-1334263.1 0.271 0.015 AD-1334315.1 0.744 0.043 AD-1334261.1 0.689 0.021 AD-1334289.1 0.577 0.013 AD-1334288.1 0.276 0.010 AD-1334256.1 0.339 0.008 AD-1334255.1 0.390 0.026 AD-1334287.1 0.857 0.091 AD-1334253.1 0.808 0.073 AD-1334286.1 0.328 0.010 AD-1334285.1 0.636 0.045 AD-1334284.1 0.600 0.057 AD-1334279.1 0.249 0.013 AD-1334241.1 0.266 0.075 AD-1334276.1 0.335 0.021 AD-1334275.1 0.329 0.044 AD-1334271.1 0.381 0.034 AD-1334317.1 0.245 0.023 AD-1334316.1 0.467 0.044 AD-1334314.1 0.368 0.022 AD-1334313.1 0.266 0.023 AD-1334312.1 0.472 0.040 AD-1334311.1 0.320 0.051 AD-1334247.1 0.467 0.010 AD-1334246.1 0.375 0.022 AD-1334308.1 0.284 0.015 AD-1334307.1 0.702 0.045 AD-1334306.1 0.291 0.065 AD-1334305.1 0.308 0.054 AD-1334304.1 0.275 0.036 AD-1334303.1 0.430 0.110 AD-1334301.1 0.319 0.050 AD-1334297.1 0.433 0.019 AD-1334295.1 0.697 0.041 AD-1334292.1 0.303 0.041 AD-1334291.1 0.397 0.080 AD-1334290.1 0.359 0.049 AD-1334262.1 0.559 0.041 AD-1334283.1 0.280 0.025 AD-1334282.1 0.330 0.063 AD-1334281.1 0.261 0.011 AD-1334280.1 0.268 0.010 AD-1334260.1 0.550 0.036 AD-1334259.1 0.223 0.031 AD-1334258.1 0.812 0.055 AD-1334257.1 0.454 0.035 AD-1334254.1 0.486 0.029 AD-1334252.1 0.372 0.138 AD-1334251.1 0.817 0.229 AD-1334244.1 0.302 0.051 AD-1334243.1 0.287 0.024 AD-1334238.1 0.675 0.159 AD-1334237.1 0.693 0.030 AD-1334235.1 0.427 0.063 AD-1334233.1 0.300 0.010 AD-1334232.1 0.490 0.072 AD-1334231.1 0.472 0.041 AD-1334230.1 0.280 0.060 AD-1334229.1 0.355 0.039 AD-1334228.1 0.394 0.061 AD-1334227.1 0.226 0.043 AD-1334226.1 0.714 0.146 AD-1334225.1 0.230 0.051 AD-1334224.1 0.491 0.086 AD-1334223.1 0.273 0.019 AD-1334222.1 0.493 0.180 AD-1334221.1 0.343 0.027 AD-1334220.1 0.289 0.027 AD-1334219.1 0.349 0.055 AD-1334218.1 0.642 0.051 AD-1334217.1 0.998 0.116 AD-1334216.1 0.479 0.048 AD-1334215.1 0.543 0.092 AD-1334214.1 1.007 0.023 AD-1334213.1 0.218 0.049 AD-1334212.1 0.282 0.025 AD-1334211.1 0.645 0.070 AD-1334209.1 0.654 0.054 AD-1334208.1 0.886 0.078 AD-1334207.1 0.190 0.012 AD-1334206.1 0.364 0.083 AD-1334205.1 0.692 0.006 AD-1334204.1 1.004 0.142 AD-1334203.1 0.780 0.160 AD-1334202.1 1.006 0.043 AD-1334201.1 0.303 0.030 AD-1334200.1 0.331 0.036 AD-1334199.1 0.790 0.139 AD-1334198.1 0.270 0.094 AD-1334197.1 0.810 0.027 AD-1334196.1 0.550 0.026 AD-1334195.1 0.570 0.038 AD-1334194.1 0.321 0.094 AD-1334193.1 0.571 0.065 AD-1334192.1 0.729 0.096 AD-1334191.1 0.422 0.021 AD-1334190.1 0.917 0.107 AD-1334189.1 0.347 0.048 AD-1334188.1 0.699 0.171 AD-1334187.1 0.560 0.058 AD-1334186.1 0.230 0.015 AD-1334185.1 0.551 0.109 AD-1334184.1 0.540 0.013 AD-1334183.1 0.286 0.024 AD-1334182.1 0.431 0.041 AD-1334181.1 0.545 0.067 AD-1334180.1 0.509 0.068 AD-1334179.1 0.935 0.225 AD-1334178.1 0.303 0.053 AD-1334177.1 0.821 0.170 AD-1334176.1 0.744 0.065 AD-1334175.1 1.290 0.351 AD-1334174.1 0.984 0.581 AD-1334173.1 0.428 0.120 AD-1334172.1 0.549 0.068 AD-1334171.1 0.468 0.124 AD-1334170.1 0.505 0.068 AD-1334169.1 0.249 0.026 AD-1334168.1 0.213 0.026 AD-1334167.1 0.259 0.032 AD-1334166.1 0.694 0.442 AD-1334165.1 0.324 0.106 AD-1334164.1 0.391 0.046 AD-1334163.1 0.480 0.067 AD-1334162.1 0.734 0.214 AD-1334161.1 0.210 0.025 AD-1334160.1 0.642 0.031 AD-1334159.1 0.545 0.027 AD-1334158.1 0.439 0.015 AD-1334157.1 0.425 0.026 AD-1334156.1 0.442 0.024 AD-1334155.1 0.448 0.043 AD-1334154.1 0.450 0.020 AD-1334153.1 0.636 0.080 AD-1334152.1 0.483 0.059 AD-1334151.1 0.313 0.016 AD-1334150.1 0.604 0.066 AD-1334149.1 0.539 0.095 AD-1334148.1 0.370 0.008 AD-1334147.1 0.332 0.039 AD-1334146.1 0.262 0.043 AD-1334145.1 0.903 0.041 AD-1334144.1 0.912 0.168 AD-1334143.1 0.330 0.033 AD-1334142.1 0.825 0.021 AD-1334141.1 0.493 0.058 AD-1334140.1 0.528 0.012 AD-1334139.1 0.946 0.113 AD-1334138.1 0.439 0.101 AD-1334136.1 0.423 0.078 AD-1334135.1 0.309 0.109 AD-1334134.1 0.311 0.030 AD-1334133.1 0.712 0.147 AD-1334132.1 0.485 0.153 AD-1334131.1 0.160 0.085 AD-1334130.1 0.516 0.028 AD-1334129.1 0.496 0.030 AD-1334128.1 0.378 0.020 AD-1334127.1 0.422 0.028 AD-1334126.1 0.586 0.037 AD-1334125.1 0.731 0.098 AD-1334124.1 0.224 0.052 AD-1334123.1 0.286 0.066 AD-1334122.1 0.801 0.064 AD-1334121.1 1.017 0.095 AD-1334120.1 0.424 0.052 AD-1334119.1 0.578 0.056 AD-1334118.1 0.573 0.014 AD-1334117.1 0.500 0.034 AD-1334116.1 0.394 0.009 AD-1334115.1 0.455 0.047 AD-1334114.1 0.949 0.041 AD-1334113.1 0.928 0.090 AD-1334112.1 0.883 0.147 AD-1334111.1 0.467 0.017 AD-1334110.1 0.443 0.027 AD-1334109.1 0.438 0.042 AD-1334108.1 0.243 0.019 AD-1334107.1 0.477 0.074 AD-1334106.1 0.755 0.048 AD-1334105.1 0.788 0.062 AD-1334104.1 0.502 0.078 AD-1334103.1 0.317 0.042 AD-1334102.1 0.282 0.051 AD-1334101.1 0.321 0.054 AD-1334100.1 0.679 0.029 AD-1334099.1 0.196 0.061 AD-1334098.1 0.683 0.086 AD-1334097.1 0.860 0.127

Example 3. In Vivo Screening of dsRNA Duplexes in Mice

siRNA molecules targeting the MUC5B gene, identified from the above in vitro studies, are evaluated in vivo.

For example, the siRNA molecules may be assessed for theit ability to decrease MUC5B expression in a transgenic mouse overexpressing human MUC5b. Alternatively or in addition, suitable animal models of lung diseases may be used. Some examples of available models of pulmonary fibrosis include the bleomycin mouse model of pulmonary fibrosis (Muggia F M, et al. (1983). Cancer Treat Rev 10: 221-243); the FITC-induced model for pulmonary fibrosis (Roberts S N, et al. (1995). J Pathol 176: 309-318); irradiation-induced pulmonary fibrosis mice (Rube C E, et al. (2000) Int J Radiat Oncol Biol Phys 47: 1033-1042); and instillation of mineral fibers into the rodent lung (Davis G S, et al. (1998) J Environ Pathol Toxicol Oncol 17: 81-97). Exemplary animal models of cystic fibrosis include the CFTR knockout mouse models (Semaniakou A, et al. Front Pharmacol. 2018; 9: 1475). Some examples of animal models for chronic obstructive pulmonary disease (COPD) include cigarette/smoke-induced model for COPD, and lipopolysaccharide (LPS)-induced model for COPD (Ghorani, V et al, Tob Induc Dis. 2017; 15: 25). Many of the mouse models are commercially available from the Jackson Laboratory or Chrales River.

Selected dsRNA agents designed and assayed in Example 1 are assessed for their ability to reduce the level of MUC5B expression in these animal models and to clear pulmonary fibrosis or mucus buildup in the lung.

Mice are administered, via pulmonary or subcutaneous delivery, a dsRNA molecule at a dose of 0.1 mg/kg, 1 mg/kg or 10 mg/kg. Uptake of dsRNA in bronchioles and alveoli and expression level of MUC5B in whole lung of treated mice is measured. Expression levels of MUC5B are further evaluated by in situ hybridization in mice bronchus and bronchiole.

Example 4. In Vitro Analysis of Duplexes Targeting MUC5B

Additional duplexes targeting mouse MUC5B (mouse NCBI refseqID: NM_028801.2; NCBI GeneID: 74180) were designed and synthesized as described above. The mouse NM_028801.2 REFSEQ mRNA has a length of 14963 bases.

A detailed list of a set of the unmodified dsRNA sense and antisense strand sequences targeting MUC5B is shown in Table 9.

A detailed list of a set of the modified dsRNA sense and antisense strand sequences targeting MUC5B is shown in Table 10.

These duplexes were assessed for activity in single dose screens using the dual luciferase screening assay described above.

Briefly, Cos7 cells were transfected by adding 50 μL of dsRNA duplexes and 75 ng per well of mouse MUC5B plasmid encoding a portion of the murine MUC5B gene targeted by the duplex being assessed along with 100 μL of Opti-MEM plus 0.5 μL of Lipofectamine 2000 per well (Invitrogen, Carlsbad CA. cat #13778-150) and then incubated at room temperature for 15 minutes. The mixture was then added to the cells which are re-suspended in 35 μL of fresh complete media. The transfected cells were incubated at 37° C. in an atmosphere of 5% CO2. Single-dose experiments were performed at 10 nM, 1.0 nM, and 0.1 nM.

Twenty-four hours after the siRNAs and psiCHECK2 plasmid were transfected, Firefly (transfection control) and Renilla (fused to MUC5B target sequence) luciferase were measured. First, media was removed from cells. Then Firefly luciferase activity was measured by adding 75 μL of Dual-Glo® Luciferase Reagent equal to the culture medium volume to each well and mixing. The mixture was incubated at room temperature for 30 minutes before luminescense (500 nm) was measured on a Spectramax (Molecular Devices) to detect the Firefly luciferase signal. Renilla luciferase activity was measured by adding 75 μL of room temperature of Dual-Glo® Stop & Glo® Reagent to each well and the plates were incubated for 10-15 minutes before luminescence was again measured to determine the Renilla luciferase signal. The Dual-Glo® Stop & Glo® Reagent quenches the firefly luciferase signal and sustained luminescence for the Renilla luciferase reaction. siRNA activity was determined by normalizing the Renilla (MUC5B) signal to the Firefly (control) signal within each well. The magnitude of siRNA activity was then assessed relative to cells that were transfected with the same vector but were not treated with siRNA or were treated with a non-targeting siRNA. All transfections were done with n=4.

The results of these assays are shown in Table 11 and in FIG. 1 .

TABLE 9 Unmodified Sense and Antisense Strand MUC5B dsRNA Sequences SEQ Range in SEQ Range in Duplex Sense Sequence ID NM_ Antisense Sequence ID NM_ Name 5′ to 3′ NO: 028801.2 5′ to 3′ NO: 028801.2 AD- CCAGCUGUCAGUGAGUUCUAA 4192 697-717 UUAGAACUCACUGACAGCUGGGA 4214 695-717 1311859.1 AD- UGAACUCCAUCUUUACGCAGA 4193 1562-1582 UCUGCGUAAAGAUGGAGUUCAGG 4215 1560-1582 1312581.1 AD- AUCCUUCUUCAUCAUAGUACA 4194 1623-1643 UGUACUAUGAUGAAGAAGGAUGA 4216 1621-1643 1312642.1 AD- GGUUGACUCUACAAAAUACUA 4195 3360-3380 UAGUAUUUUGUAGAGUCAACCUG 4217 3358-3380 1314054.1 AD- UGACUCUACAAAAUACUAUGA 4196 3363-3383 UCAUAGUAUUUUGUAGAGUCAAC 4218 3361-3383 1314057.1 AD- ACUCUACAAAAUACUAUGAAA 4197 3365-3385 UUUCAUAGUAUUUUGUAGAGUCA 4219 3363-3385 1314059.1 AD- CUCUACAAAAUACUAUGAAGA 4198 3366-3386 UCUUCAUAGUAUUUUGUAGAGUC 4220 3364-3386 1314060.1 AD- UCACUUCAACUUCUAUGUCGA 4199 4553-4573 UCGACAUAGAAGUUGAAGUGAGU 4221 4551-4573 1315057.1 AD- AGUCUCUCUACCUUUCCAGCA 4200 8602-8622 UGCUGGAAAGGUAGAGAGACUAG 4222 8600-8622 1316271.1 AD- GGUCUCAACGACAGAAACUGA 4201 5079-5099 UCAGUUUCUGUCGUUGAGACCGA 4223 5077-5099 1316856.1 AD- CCUCUUCCCAACACUAGUCUA 4202 10549-10569 UAGACUAGUGUUGGGAAGAGGCA 4224 10547-10569 1317692.1 AD- GUGCUUUAACUACAAUAUACA 4203 11154-11174 UGUAUAUUGUAGUUAAAGCACAU 4225 11152-11174 1318239.1 AD- UCCACCUUUCAGACUAACCGA 4204 11278-11298 UCGGUUAGUCUGAAAGGUGGACU 4226 11276-11298 1318336.1 AD- CCACCUUUCAGACUAACCGUA 4205 11279-11299 UACGGUUAGUCUGAAAGGUGGAC 4227 11277-11299 1318337.1 AD- CACCUUUCAGACUAACCGUCA 4206 11280-11300 UGACGGUUAGUCUGAAAGGUGGA 4228 11278-11300 1318338.1 AD- GGAUAUCAUCUACAAUAAGAA 4207 12054-12074 UUCUUAUUGUAGAUGAUAUCCCC 4229 12052-12074 1318879.1 AD- CUCUACCACUGUACCUUUGCA 4208 12171-12191 UGCAAAGGUACAGUGGUAGAGGA 4230 12169-12191 1318976.1 AD- CUACCACUGUACCUUUGCCUA 4209 12173-12193 UAGGCAAAGGUACAGUGGUAGAG 4231 12171-12193 1318978.1 AD- GGCAACAAUCAAAUCAUUCUA 4210 12283-12303 UAGAAUGAUUUGAUUGUUGCCCU 4232 12281-12303 1319047.1 AD- UGCCACUUCCACUACGAGUGA 4211 12379-12399 UCACUCGUAGUGGAAGUGGCAAG 4233 12377-12399 1319143.1 AD- CACUUCCACUACGAGUGUGAA 4212 12382-12402 UUCACACUCGUAGUGGAAGUGGC 4234 12380-12402 1319146.1 AD- CCAGAUGCCUUCUUCAGAAGA 4213 13105-13125 UCUUCUGAAGAAGGCAUCUGGAG 4235 13103-13125 1319731.1

TABLE 10 Modified Sense and Antisense Strand MUC5B dsRNA Sequences SEQ Antisense SEQ SEQ Duplex Sense Sequence ID Sequence ID mRNA Target ID Name 5′ to 3′ NO: 5′ to 3′ NO: Sequence NO: AD- cscsagc(Uhd)Gfu 4236 VPusUfsagaAfcUf 4258 UCCCAGCUGUCAGUG 4280 1311859.1 CfAfGfugaguucus CfacugAfcAfgcug AGUUCUAC asa gsgsa AD- usgsaac(Uhd)Cfc 4237 VPusCfsugcGfuAf 4259 CCUGAACUCCAUCUU 4281 1312581.1 AfUfCfuuuacgcas AfagauGfgAfguuc UACGCAGA gsa asgsg AD- asusccu(Uhd)Cfu 4238 VPusGfsuacUfaUf 4260 UCAUCCUUCUUCAUC 4282 1312642.1 UfCfAfucauaguas GfaugaAfgAfagga AUAGUACA csa usgsa AD- gsgsuug(Ahd)Cfu 4239 VPusAfsguaUfuUf 4261 CAGGUUGACUCUACA 4283 1314054.1 CfUfAfcaaaauacs UfguagAfgUfcaac AAAUACUA usa csusg AD- usgsacu(Chd)Ufa 4240 VPusCfsauaGfuAf 4262 GUUGACUCUACAAAA 4284 1314057.1 CfAfAfaauacuaus UfuuugUfaGfaguc UACUAUGA gsa asasc AD- ascsucu(Ahd)Cfa 4241 VPusUfsucaUfaGf 4263 UGACUCUACAAAAUA 4285 1314059.1 AfAfAfuacuaugas UfauuuUfgUfagag CUAUGAAG asa uscsa AD- csuscua(Chd)Afa 4242 VPusCfsuucAfuAf 4264 GACUCUACAAAAUAC 4286 1314060.1 AfAfUfacuaugaas GfuauuUfuGfuaga UAUGAAGC gsa gsusc AD- uscsacu(Uhd)Cfa 4243 VPusCfsgacAfuAf 4265 ACUCACUUCAACUUC 4287 1315057.1 AfCfUfucuaugucs GfaaguUfgAfagug UAUGUCGU gsa asgsu AD- asgsucu(Chd)Ufc 4244 VPusGfscugGfaAf 4266 CUAGUCUCUCUACCU 4288 1316271.1 UfAfCfcuuuccags AfgguaGfaGfagac UUCCAGCC csa usasg AD- gsgsucu(Chd)Afa 4245 VPusCfsaguUfuCf 4267 UCGGUCUCAACGACA 4289 1316856.1 CfGfAfcagaaacus UfgucgUfuGfagac GAAACUGC gsa csgsa AD- cscsucu(Uhd)Cfc 4246 VPusAfsgacUfaGf 4268 UGCCUCUUCCCAACA 4290 1317692.1 CfAfAfcacuagucs UfguugGfgAfagag CUAGUCUG usa gscsa AD- gsusgcu(Uhd)Ufa 4247 VPusGfsuauAfuUf 4269 AUGUGCUUUAACUAC 4291 1318239.1 AfCfUfacaauauas GfuaguUfaAfagca AAUAUACG csa csasu AD- uscscac(Chd)Ufu 4248 VPusCfsgguUfaGf 4270 AGUCCACCUUUCAGA 4292 1318336.1 UfCfAfgacuaaccs UfcugaAfaGfgugg CUAACCGU gsa ascsu AD- cscsacc(Uhd)Ufu 4249 VPusAfscggUfuAf 4271 GUCCACCUUUCAGAC 4293 1318337.1 CfAfGfacuaaccgs GfucugAfaAfggug UAACCGUC usa gsasc AD- csasccu(Uhd)Ufc 4250 VPusGfsacgGfuUf 4272 UCCACCUUUCAGACU 4294 1318338.1 AfGfAfcuaaccgus AfgucuGfaAfaggu AACCGUCC csa gsgsa AD- gsgsaua(Uhd)Cfa 4251 VPusUfscuuAfuUf 4273 GGGGAUAUCAUCUAC 4295 1318879.1 UfCfUfacaauaags GfuagaUfgAfuauc AAUAAGAC asa csCSC AD- csuscua(Chd)Cfa 4252 VPusGfscaaAfgGf 4274 UCCUCUACCACUGUA 4296 1318976.1 CfUfGfuaccuuugs UfacagUfgGfuaga CCUUUGCC csa gsgsa AD- csusacc(Ahd)Cfu 4253 VPusAfsggcAfaAf 4275 CUCUACCACUGUACC 4297 1318978.1 GfUfAfccuuugccs GfguacAfgUfggua UUUGCCUC usa gsasg AD- gsgscaa(Chd)Afa 4254 VPusAfsgaaUfgAf 4276 AGGGCAACAAUCAAA 4298 1319047.1 UfCfAfaaucauucs UfuugaUfuGfuugc UCAUUCUC usa cscsu AD- usgscca(Chd)Ufu 4255 VPusCfsacuCfgUf 4277 CUUGCCACUUCCACU 4299 1319143.1 CfCfAfcuacgagus AfguggAfaGfuggc ACGAGUGU gsa asasg AD- csascuu(Chd)Cfa 4256 VPusUfscacAfcUf 4278 GCCACUUCCACUACG 4300 1319146.1 CfUfAfcgagugugs CfguagUfgGfaagu AGUGUGAA asa gsgsc AD- cscsaga(Uhd)Gfc 4257 VPusCfsuucUfgAf 4279 CUCCAGAUGCCUUCU 4301 1319731.1 CfUfUfcuucagaas AfgaagGfcAfucug UCAGAAGC gsa gsasg

TABLE 11 MUC5B Single Dose In Vitro Screen in Cos 7 Cells. Duplex Name 10 nM stdev 1 nM stdev 0.1 nM stdev AD-1311859.1 19.1 2.9 31.2 2.9 64.0 2.8 AD-1312581.1 46.7 2.6 64.3 3.8 97.4 4.8 AD-1312642.1 36.3 2.1 46.0 4.1 72.1 5.3 AD-1314054.1 14.9 1.7 26.4 2.9 64.0 3.5 AD-1314057.1 19.7 2.4 41.3 4.5 74.1 7.0 AD-1314059.1 25.2 2.5 36.5 4.7 74.7 5.7 AD-1314060.1 23.6 3.4 51.8 6.3 85.1 9.9 AD-1315057.1 20.2 2.3 43.5 2.5 77.5 5.9 AD-1316856.1 27.5 2.2 41.6 1.4 73.0 6.7 AD-1316271.1 9.3 1.9 14.4 4.0 44.1 7.2 AD-1317692.1 16.4 2.6 31.6 3.5 64.3 2.0 AD-1318239.1 4.7 1.4 8.4 2.5 20.5 5.4 AD-1318336.1 13.6 1.2 51.3 21.6 64.4 3.2 AD-1318337.1 7.6 1.4 18.3 3.8 52.0 4.9 AD-1318338.1 11.6 0.9 22.5 3.9 59.1 7.0 AD-1318879.1 79.8 9.0 99.2 3.2 101.9 3.5 AD-1318976.1 13.7 2.3 40.0 3.6 74.7 0.9 AD-1318978.1 32.7 3.1 48.7 2.6 85.6 10.7 AD-1319047.1 26.2 3.2 37.3 3.4 66.4 6.1 AD-1319143.1 60.6 9.6 71.0 3.4 96.3 7.6 AD-1319146.1 77.7 8.7 89.9 6.0 98.4 5.3 AD-1319731.1 14.3 2.6 38.0 5.6 65.0 4.2

Example 5. In Vivo Screening of dsRNA Duplexes in Mice

A subset of the duplexes in Example 4, above, were assessed in vivo in 7 week old C57BL/6N female mice. Mice, n=4, were administered a single 10 mg/kg dose of AD-1318337, AD-1318338, AD-1314054, AD-1317692, or AD-1318239, or saline control by orotracheal application on Day 0. On Day 10, animals were sacrificed, whole lung samples were harvested, and the level of MUC5B mRNA in the sample was quantified as described above. The results of these assays are shown in FIG. 2 and demonstrate that orotracheal application of the indicated dsRNA agents can effectively inhibit MUC5B expression in vivo.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments and methods described herein. Such equivalents are intended to be encompassed by the scope of the following claims. 

1. A double stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Mucin 5B (MUC5B) in a cell, (a) wherein the dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, wherein the sense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of a portion of the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:1, and the antisense strand comprises a nucleotide sequence comprising at least 15 contiguous nucleotides, with 0, 1, 2, or 3 mismatches, of the corresponding portion of the nucleotide sequence of SEQ ID NO:6, or a nucleotide sequence having at least 90% nucleotide sequence identity to a portion of the nucleotide sequence of SEQ ID NO:6; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties; or (b) wherein the antisense strand comprises a region complementary to part of an mRNA encoding a MUC5B gene (SEQ ID NO:1), wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties; or (c) wherein the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense nucleotide sequences in any one of Tables 2-7, 9, and 10, wherein each strand independently is 14 to 30 nucleotides in length; and wherein the sense strand or the antisense strand is conjugated to one or more lipophilic moieties.
 2. (canceled)
 3. (canceled)
 4. The dsRNA agent of claim 1, wherein the sense strand or the antisense strand is a sense strand or an antisense strand selected from the group consisting of any of the sense strands and antisense strands in any one of Tables 2-7, 9, and
 10. 5.-10. (canceled)
 11. The dsRNA agent of claim 1, wherein the dsRNA agent comprises at least one modified nucleotide.
 12. (canceled)
 13. (canceled)
 14. The dsRNA agent of claim 11, wherein at least one of the modified nucleotides is selected from the group a deoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a 2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide, a 2′-methoxyethyl modified nucleotide, a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, a non-natural base comprising nucleotide, a tetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modified nucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, a nucleotide comprising a 5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′ phosphate mimic, a nucleotide comprising vinyl phosphonate, a nucleotide comprising adenosine-glycol nucleic acid (GNA), a nucleotide comprising thymidine-glycol nucleic acid (GNA)S-Isomer, a nucleotide comprising 2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising 2′-deoxythymidine-3′phosphate, a nucleotide comprising 2′-deoxyguanosine-3′-phosphate, a 2′-O hexadecyl nucleotide, a nucleotide comprising a 2′-phosphate, a cytidine-2′-phosphate nucleotide, a guanosine-2′-phosphate nucleotide, a 2′-O-hexadecyl-cytidine-3′-phosphate nucleotide, a 2′-O-hexadecyl-adenosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-guanosine-3′-phosphate nucleotide, a 2′-O-hexadecyl-uridine-3′-phosphate nucleotide, a a 5′-vinyl phosphonate (VP), a 2′-deoxyadenosine-3′-phosphate nucleotide, a 2′-deoxycytidine-3′-phosphate nucleotide, a 2′-deoxyguanosine-3′-phosphate nucleotide, a 2′-deoxythymidine-3′-phosphate nucleotide, a 2′-deoxyuridine nucleotide, and a terminal nucleotide linked to a cholesteryl derivative and a dodecanoic acid bisdecylamide group; and combinations thereof. 15.-17. (canceled)
 18. The dsRNA agent of claim 14, further comprising at least one phosphorothioate internucleotide linkage.
 19. (canceled)
 20. The dsRNA agent of claim 1, wherein each strand is no more than 30 nucleotides in length. 21.-31. (canceled)
 32. The dsRNA agent of claim 1, wherein one or more lipophilic moieties are conjugated to one or more internal positions on at least one strand. 33.-41. (canceled)
 42. The dsRNA agent of claim 1, wherein the one or more lipophilic moieties are conjugated to one or more of the internal positions selected from the group consisting of positions 4-8 and 13-18 on the sense strand, and positions 6-10 and on the antisense strand, counting from the 5′-end of each strand. 43.-51. (canceled)
 52. The dsRNA agent of claim 1, wherein the lipophilic moiety contains a saturated or unsaturated C4-C30 hydrocarbon chain, and an optional functional group selected from the group consisting of hydroxyl, amine, carboxylic acid, sulfonate, phosphate, thiol, azide, and alkyne. 53.-68. (canceled)
 69. The dsRNA agent of claim 1, further comprising a phosphate or phosphate mimic at the 5′-end of the antisense strand. 70.-72. (canceled)
 73. An isolated cell containing the dsRNA agent of claim
 1. 74. A pharmaceutical composition for inhibiting expression of a MUC5B gene, comprising the dsRNA agent of claim
 1. 75. (canceled)
 76. A device for oral inhalative administration comprising the dsRNA agent of claim
 1. 77. (canceled)
 78. An in vitro method of inhibiting expression of a MUC5B gene in a cell, the method comprising: (a) contacting the cell with the dsRNA agent of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the MUC5B gene, thereby inhibiting expression of the MUC5B gene in the cell.
 79. (canceled)
 80. (canceled)
 81. The method of claim 76, wherein the expression of the MUC5B gene is inhibited by at least 50%.
 82. A method of treating a subject having a Mucin 5B- (MUC5B-) associated disease or a subject at risk of developing a MUC5B-associated disease, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby treating said subject.
 83. The method of claim 82, wherein the subject is a human.
 84. The method of claim 82, wherein the MUC5B-associated disease is pulmonary fibrosis, cystic fibrosis, or chronic obstructive pulmonary disease (COPD).
 85. (canceled)
 86. (canceled)
 87. The method of claim 82, wherein the dsRNA agent is administered to the subject at a dose of about 0.01 mg/kg to about 50 mg/kg. 88.-90. (canceled)
 91. The method of claim 82, further comprising administering to the subject an additional agent or a therapy suitable for treatment or prevention of a MUC5B-associated disorder. 92.-95. (canceled) 